Abstract
As an efficient fungus in secreting cellulase, Trichoderma orientalis EU7-22 has an important value in the degradation of biomass. Compared with other filamentous fungi, it has an important, unique nature, and it deserves intensive study. Therefore, the function of positive transcriptional regulator ACE3 was investigated for cellulase and hemicellulase production in the strain. As the ace3 knockout strain, the Δace3 mutant was constructed by homologous recombination, so that the enzyme activities (FPA, CMC, CBH, BG, XYN) and the extracellular protein concentration in the mutant strain decreased to 17.8%, 8.3%, 0.14%, 1.8%, 6.8%, and 19.2% of the parent strain, respectively. The transcription level of cellulase and hemicellulase genes also decreased significantly. The result of SDS-PAGE demonstrated that the Δace3 mutant only clearly showed a protein band, which was characterized by protein profiling with LC-MS/MS and identified as the GH10 family of xylanases. It was proposed that ACE3 is a main up-regulation transcriptional factor of T. orientalis EU7-22 and expected to be applicable to further genetic modification.
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ACE3 as a Master Transcriptional Factor Regulates Cellulase and Xylanase Production in Trichoderma orientalis EU7-22
Fuchuan Liu, Yong Xue, Jian Liu,* Lihui Gan, and Minnan Long *
As an efficient fungus in secreting cellulase, Trichoderma orientalis EU7-22 has an important value in the degradation of biomass. Compared with other filamentous fungi, it has an important, unique nature, and it deserves intensive study. Therefore, the function of positive transcriptional regulator ACE3 was investigated for cellulase and hemicellulase production in the strain. As the ace3 knockout strain, the Δace3 mutant was constructed by homologous recombination, so that the enzyme activities (FPA, CMC, CBH, BG, XYN) and the extracellular protein concentration in the mutant strain decreased to 17.8%, 8.3%, 0.14%, 1.8%, 6.8%, and 19.2% of the parent strain, respectively. The transcription level of cellulase and hemicellulase genes also decreased significantly. The result of SDS-PAGE demonstrated that the Δace3 mutant only clearly showed a protein band, which was characterized by protein profiling with LC-MS/MS and identified as the GH10 family of xylanases. It was proposed that ACE3 is a main up-regulation transcriptional factor of T. orientalis EU7-22 and expected to be applicable to further genetic modification.
Keywords: Trichoderma orientalis EU7-22; Transcriptional factor ACE3; Cellulase; Xylanase
Contact information: College of Energy, Xiamen University, Xiamen 361005, P. R. China;
*Corresponding authors: jianliu@xmu.edu.cn (Jian Liu); longmn@xmu.edu.cn (Minnan Long)
INTRODUCTION
Lignocellulose is the most abundant renewable resource in the world, and microbial hydrolytic enzymes, especially those from different species of fungi, play the main role in the degradation of lignocellulosic biomass. However, currently only limited strains such as Trichoderma reesei and Aspergillus spp. of ascomycetes have been developed for industrial application (Pariza and Johnson 2001). Trichoderma orientalis EU7-22 was obtained by mutation screening. Some articles report that compared with T. reesei, T. koningii, A. niger, and P. decumbens, T. orientalis EU7-22 shows a higher activity of cellulase (exo-glucanase, endo-glucanase, and β-glucosidase) and xylanase, and the corresponding genes have also been cloned and classified (Long et al. 2012, 2013). Compared with other filamentous fungi, it has an important unique nature, and it deserves intensive study (Long et al. 2012, 2013; Li et al. 2014). This is meaningful for exploiting the mechanism of enzyme production in EU7-22.
Transcription factors play a crucial role in the regulation of cellulase and xylanase gene expression in filamentous fungi. Cre1/A acts as a repressor for mediating carbon catabolite repression (CCR), and is conserved through the fungal kingdom (Todd et al. 2014). XlnR in Aspergillus niger encodes a binuclear DNA cluster binding protein that promotes the expression of cellulase and xylanase genes (Hasper et al. 2004). Its orthologs have also been identified in Talaromyces cellulolyticus, Aspergillus oryzae (xlnR), and T. reesei (xyr1) (Stricker et al. 2006; Noguchi et al. 2011; Fujii et al. 2014). Recently, Häkkinen et al. (2014) conducted a preliminary study on ACE3 acting as a novel positive regulator in T. reesei, but further extending research is necessary. Through phylogenetic analysis (Fig. 1), this transcription factor spreads throughout the whole fungi kingdom, and its function has not been characterized in other species (Benocci et al. 2017). In this study, the transcription factor ACE3 was cloned and sequenced in the Trichoderma orientalis EU7-22. The cellulase and xylanase activity, transcription level, and protein production in the Δace3 mutant are all investigated to compare with the parent strain, which contributes to identifying the function of ACE3 in this strain, and further demonstrating the role of ACE3 factor during the degradation of cellulase and hemicellulase in filamentous fungi.
EXPERIMENTAL
Strain and Culture Conditions
Trichoderma orientalis EU7-22 was preserved by the authors’ laboratory and used as the parent strain in this study. The strain was cultivated on potato dextrose agar (PDA) for 4 days to 6 days, and then the spores were collected and diluted to 1×105 spores/mL with sterile water. For DNA extraction and enzyme production, the spores were inoculated in minimum medium (2 g/L glucose, 15 g/L KH2PO4, 5 g/L (NH4)2SO4, 0.6 g/L CaCl2, 0.6 g/L MgSO4, and pH 5.5) for 18 h at 30 ºC with 180 rpm agitation on a rotary shaker, then inoculated as 10% inoculum to inducing medium (1% Avicel®, 1% wheat bran, 0.05% CaCl2, 0.5% peptone, 0.25% KH2PO4, 0.05% MgSO4, and 0.4% Tween-80).
Cloning of ACE3, Upstream and Downstream Sequences
According to ace3 of Trichoderma virens (GenBank accession No. XM014102116.1), T. reesei (GenBank accession No. CP016233.1), and Purpureocillium lilacinum (GenBank accession No. XM018319611.1), degenerate primers ACE3-F1 and ACE3-R2 were designed in the conserved sequence and used for amplifying the homologous ace3 gene of T. orientalis EU7-22. The sequences were procured from the NCBI database (https://www.ncbi.nlm.nih.gov/genbank/). After purification, the product was linked to the vector of pMD 19-T. Escherichia coliDH5ɑ was used as the host of vector propagation. The gene was sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). In the same way, the primers (listed in the supplementary material) used for amplifying the upstream and downstream of ace3 were designed according to conserved sequences. After a total of three times of PCR using EU7-22 genome DNA as the template, the products were sequenced and then spliced by DNAMAN. Finally, the authors got the ace3 gene, including the flanking region. The intron was analyzed by DNAMAN, and the exon was translated into amino acid sequences using Primer Premier 5.0. A phylogenetic tree was constructed based on MEGA 6.06 software by neighbor-joining (NJ) method. BlASTP in NCBI was used to analyze the amino acid sequences and orthologs found in different species of filamentous fungi family.
Construction of the ΔACE3 Mutant
The ace3 gene was knocked out by homologous recombination, and the selective marker hph was used for isolating the transformants. As the sketch map showed in Fig. 2a, the deletion cassette Δace3::hph was constructed using primers P2 and P5 by fusion PCR with three fragments (5’ ace3 flanking region, the hygromycin B resistance gene hph expression cassette, and 3’ ace3 flanking region) (Szewczyk et al. 2006). The Δace3::hph cassette was amplified and transformed into the wild-type protoplasts to obtain the Δace3 mutant. The transformants were isolated using minimum medium plates supplemented with 1% hygromycin B. The primers used for the construction were listed in the Table 1.
Enzymatic and Protein Concentration Assays
For the enzyme assays, the spores were incubated the same way as in the previous research (Long et al. 2018). Filter paper activity (FPA) and endoglucanase activity (CMC) were measured using the reported method (Ghose 1987). pNPC and pNPG (SIGMA, St. Louis, MO, USA) were used as the substrates to determine cellobiohydrolase activity (CBH) and β-glucosidase activity (BG), respectively (Saha 2004). Xylanase activity (XYN) was examined with the substrate of xylan (extracted from sugarcane fibre) (Bailey and Jeger 1992). The protein concentration was measured by the Bradford protein assay kit (Sangon, China). Under the assayed conditions, the amount of enzyme required to release 1 μmol of product (glucose/xylose/p-nitrophenyl) from the substrate per min was defined as one unit of enzyme activity.
Phenotypic Analysis
The spores were diluted to 1×107 spores/mL and spotted on the plates, and then were cultivated at 30 °C for 4 days. The colony diameter was measured every 12 h.
RNA Preparation and Quantitative Real-Time Reverse Transcription PCR
Based on the above results, a quantitative PCR was carried out for the analysis of gene transcription (cbh1, cbh2, eg1, eg2, bgl1, xyn1, and xyn2) (Long et al. 2012). The protocol for RNA extraction and RT-qPCR was shown in the previous research (Long et al. 2018). The transcription levels of target genes of T. orientalis and Δace3 mutant were normalized against that of the 18s gene.
SDS-PAGE and LC-MS/MS Analysis
The supernatant was analyzed with SDS-PAGE on 15% polyacrylamide by the method of Laemmli (1970) and stained with Coomassie Brilliant Blue R-250. The mixture of 15 μL culture supernatant and 3 μL 5× loading buffer was placed in boiling water for 10 min and was loaded into the gel. The protein band from the Δace3 mutant was excised from SDS-PAGE gels for in-gel trypsin digestion. The tryptic peptides were extracted with aqueous solution containing 67% acetonitrile/0.15% formic acid and subjected to freeze-drying. The dried peptides were dissolved in aqueous solution containing 2% acetonitrile/ 0.1% formic acid, and then separated by a fused silica capillary emitter packed with C18 resin and analyzed in an AB SCIEX TripleTOF 5600 system (Applied Biosystems, Foster City, CA, USA). Proteins were identified by the Swiss-Prot database using the Mascot program.
RESULTS AND DISCUSSION
Sequence Analysis
The sequence of ace3 was a fragment of 6241 bp, including a putative gene coding region of 2024 bp with one intron (155 bp), encoding 628 amino acids.
According to the alignment result of protein sequence from DNAMAN, the putative ACE3 in T. orientalis showed 95.55% of identity with ACE3 protein in T. reesei (XP006964947.1), and it belonged to the Zn2Cys6 class. The nucleotides and amino acids of T. orientalis EU7-22 were deposited in the GenBank with the accession No. MG720022. Through phylogenetic analysis (Fig. 1), ace3 was widely spread in filamentous fungi. Based on the conserved sequences of ace3 gene in filamentous fungi, the primers were successfully applied to clone and sequence ace3 gene, including flanking sequences in T. orientalis EU7-22.
Fig. 1. Phylogenetic tree analysis of ace3 gene homological degrees in filamentous fungi
Obtaining the Mutant
The Δace3::hph cassette was amplified and transformed into the protoplasts of EU7-22 to obtain the mutant. The transformants were isolated using minimum medium plates supplemented with 1% hygromycin B. After streak cultivation for three generations, only one transformant was obtained. From the PCR analysis with primers hph-F and hph-R, it was confirmed that the deletion cassette Δace3::hph was translated to the mutant (Fig. 2b).
Primers Cace3u-F and Cace3d-R were used for the analysis of ace3 gene, and they showed that the target gene did not exist in the mutant, but it was positive in the parent strain (Fig. 2c). Primers P1 and PgpdA-yz, as well as P6 and TrpC-yz, were used to confirm the homologous recombination location of transformant by PCR. As shown in Fig. 2d and 2e, those two fragments were not present in the parent strain, but were present in the mutant. From the above experiments, the authors got the ace3 deletion strain, named Δace3 mutant. The primers used for identifying the Δace3 mutant are listed in Table 1.
Table 1. Primers in this Study for PCR
Fig. 2. Schematic map of constructing Δace3::hph deletion cassette and identifying the Δace3 mutant. (M) maker; (1) the parent strain EU7-22; (2) the Δace3 mutant; (a) schematic map of the primers used for constructing and identifying Δace3 mutant; (b) PCR analysis of genomic DNA, EU7-22 and Δace3. Primers hph-F and hph-R test for hph gene; (c) primers Cace3u-F and Cace3u-R test for ace3 gene; (d) primers P1 and PgpdA-yz test for homologous recombination location; (e) primers TrpC-yz and P6 test for homologous recombination location
Enzyme Analysis
The parent strain T. orientalis EU7-22 and the Δace3 mutant were cultivated for enzyme activity analysis. According to Fig. 3, when ace3 gene was disrupted, the activities of cellulase and xylanase were almost lost. Comparing with EU7-22, the FPA, CMC, and XYN activities of Δace3 mutant were reduced to 17.8%, 8.3%, and 6.8%, respectively. Regarding the lack of ACE3, the CBH and BG activities were completely lost. Meanwhile, the intracellular protein concentrations were reduced to 19.2%. As shown in Fig. 3, the CMC and XYN activities still existed in the Δace3 mutant, but the CMC and XYN activities were kept at a relatively low level. It was observed that ACE3 deletion seriously repressed cellulase and xylanase activities in Trichoderma orientalis EU7-22, and the extracellular protein was also produced at a low level.
Fig. 3. Production of cellulase, xylanase activity, and protein secretion by the Δace3 mutant; (a) filter paper activity; (b) endoglucanase activity; (c) β-glucosidase activity; (d) exoglucanase activity; (e) xylanase activity; (f) protein concentration
Quantitative PCR
Based on the above results, a quantitative PCR was carried out for the analysis of cellulase and xylanase gene transcription (cbh1, cbh2, eg1, eg2, bgl1, xyn1, and xyn2). As shown in Fig. 4, after cultivating the Δace3 mutant for 48 h, the expression of cellobiohydrolase gene cbh1, cellobiohydrolase gene cbh2, endoglucanase gene eg1, and endoglucanase gene eg2 were all almost undetectable, and the expressions of β-glucosidase gene bgl1 and xylanase genes xyn1 and xyn2 were reduced to 0.06-fold, 0.02-fold, and 0.15-fold of the parent strain, respectively (Fig. 4a). In 72 h, similar results were also observed in that the transcriptions of cbh1, cbh2, eg1, and eg2 were still kept at a very low level, and those of bgl1, xyn1, and xyn2 were reduced to 0.17-fold, 0.05-fold, and 0.25-fold of the parent strain, respectively (Fig. 4b). Compared with those of 48 h, the expression levels of bgl1, xyn1, and xyn2 increased insignificantly. Eventually, the results showed that the cellulase and xylanase activities decreased rapidly as the ace3 gene was disrupted in EU7-22, and ACE3 regulated the transcription of cellulase and xylanase genes in T. orientalis EU7-22 as a major transcription factor.
Fig. 4. Analysis of the cellulase and xylanase gene expression of EU7-22 and Δace3 mutant by quantitative PCR; (a) cultivated for 48 h; (b) cultivated for 72 h
pH Value of the Supernatant
In the cultivation, pH value of supernatant from the parent strain decreased to a minimum in 24 h, but the pH value of the mutant strain showed higher pH value and increased over time. This was attributed to the disruption of ACE3, which caused the decrease of organic acid content. The color of the supernatant also became lighter after knockout (Fig. 6). The authors speculated that the transcription factor ACE3 through mediating the ambient pH, and then affected the efficiency of permeases and extracellular enzymes through mediating the ambient pH, which provides the potential application of gene regulation by acidic intermediate (Peñalva and Arst Jr. 2004).
Table 2. Primers Used for Quantitative Real Time RT-PCR
Fig. 5. pH value of T. orientalis EU7-22 and the Δace3 mutant
Fig. 6. SDS-PAGE analysis and phenotype observations of the supernatants of EU7-22 and the Δace3 mutant
Fig. 7. Phenotype observations and colony size measurements of EU7-22 and the Δace3 mutant on the different carbon plates. (A and a) 2% glucose plate; (B and b) 1% cellobiose plate; (C and c) 1% CMC-Na plate, (d) 1% Avicel® plate
SDS-PAGE and LC-MS/MS Analysis
Compared to the multiple kinds of protein in the parent strain, only one obvious protein band was detected in the SDS-PAGE of Δace3 mutant (Fig. 6). The protein was identified as part of the xylanase GH10 family (Accession No. G0RA32), but the xylanase activity was still at a very low level compared with the parent strain. The lack of ACE3 seriously influenced the secretion of proteins.
Growth Analysis of the Δace3 mutant
Cultivated in the plates using sodium carboxymethylcellulose (CMC-Na) as the sole carbon source, the phenotype and colony diameter of the Δace3 mutant significantly declined compared with the parent strain (Fig. 7C). When the carbon source was Avicel®, the Δace3 mutant produced very few spores (Fig. 7D), but a non-significant difference was found in those of glucose and cellobiose (Figs. 7A and 7B). As shown in Fig. 7c, with CMC-Na as the sole carbon resource, the colonies of Δace3 mutant were smaller than those of the parent strain. The deletion of ace3 clearly affected the colony diameter and the morphology of T. orientalis. The colonies were smaller, and they produced fewer spores and aerial hyphae than those of the parental strain in both Avicel®, and CMC-Na plates.
CONCLUSIONS
- The function of transcriptional regulator ACE3 in Trichoderma orientalis EU7-22 was identified by means of sequence analysis, enzyme analysis, phenotypic analysis, protein analysis, and quantitative PCR. ACE3 deletion seriously repressed cellulase and xylanase activities in Trichoderma orientalis, and extracellular protein was also produced at a low level. Compared with parental strains, the transcription level of the cellulase and xylanase genes (cbh1, cbh2, eg1, eg2, bgl1, xyn1, and xyn2) in the Δace3 mutant also reduced dramatically. Meanwhile, the change was related to the ambient pH of Δace3 mutant.
- SDS-PAGE coupled with LC-MS/MS was a powerful tool that was able to identify the difference in protein secretion. The only protein band in the Δace3 mutant and the same protein band in EU7-22 were characterized as part of the GH10 family of xylanases, while the xylanase activity of the Δace3 mutant was still at a low level.
- The ACE3 expression influenced the enzymatic degradation of CMC-Na and microcrystalline cellulose. Since only a few such kinds of transcription factors exist extensively, and the majority of them are limited in the subgroups of fungal species, the function investigation of ACE3 provided the reference of regulating gene expression in other fungi.
ACKNOWLEDGMENTS
This work was supported by the research fund from the Xiamen Southern Oceanographic Center (No. 4GZP59HJ29), Fujian Provincial Department of Ocean and Fisheries (No. 2015-27) and the Energy development Foundation of Energy College, Xiamen University (No. 2017NYFZ02).
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Article submitted: March 4, 2018; Peer review completed: May 13, 2018; Revised version received and accepted; July 21, 2018; Published: July 23, 2018.
DOI: 10.15376/biores.13.3.6790-6801