Enhancement of Xylanase Production by Protoplast Fusion of Trichoderma spp.

INTRODUCTION

Lignocellulosic biomass is the largest source of organic materials which its conversion into fermentable sugars conducted by holocellulase enzymes, including cellulases and hemicellulases (Andreaus et al. 2008; de Siqueira et al. 2010; Song et al. 2016). Xylanase is one of the most important hemicellulases (EC3.2.1.8) catalyzing the endohydrolysis of 1,4-b-D-xylosidic linkages in xylan, a noteworthy hemicelluloses component of lignocellulosic biomass (Kubata et al. 1994; Dashtban et al. 2009). Xylan is the second most abundant natural polysaccharide composed of β-xylopyranose residues, and its complete hydrolysis requires the action of several enzymes, including endo-1,4-β-xylanase (Polizeli et al. 2005). Xylanases have absorbed great attention due to their potential applications in the fermentation of biomass for the biofuel production, pulp and paper bleaching, improving digestibility in animal feed, juices processing in food industry and biopolishing of fabrics in textile industry (Bhat 2000; Beg et al. 2001; Harris and Ramalingam 2010; Goswami and Rawat 2015).

Fungi are the striking source of xylanases which have been extensively studied for their xylanolytic systems (Polizeli et al. 2005; van den Brink and de Vries 2011). Trichoderma strains are non-pathogenic filamentous fungi which are capable of producing a high yield of extracellular xylan degrading enzymes (Kar et al. 2006; Silva et al. 2015). Consequently, a great deal of effort is devoted to the strain improvement of Trichoderma for producing more industrial enzymes. Several studies demonstrated that genetic manipulation of fungal strains using protoplast approach could provide achieving strains with better characteristics (Dillon et al. 2008; Gong et al. 2009; Cui et al. 2014). Protoplast fusion technology can generate novel combinations of genes leading to the improvement of different characteristics of a microorganism (Patil et al. 2015). Protoplast fusion is an effective tool resulting in genetic recombination to develop superior strains in filamentous fungi (Prabavathy et al. 2006). Since Trichoderma lacks a natural mechanism for recombination of genetic material, protoplasts fusion provides an effective condition to facilitate heterokaryon formation even across species resulting in the development of interspecific and intergenetic hybrids (Savitha et al. 2010).

Holocellulases present common regulatory system in Trichoderma, although there are some differences between cellulases and xylanases gene expression regulation (Stricker et al. 2008; Kubicek et al. 2009; Seiboth et al. 2011). Due to the distribution of glycoside hydrolases encoding genes at different loci within the genome of Trichoderma, a comparative study of parents and fusants in molecular level could be informative to determine the recombination characterization (Häkkinen et al. 2012). The objective of the present study was to achieve Trichoderma strains with improved xylanase production ability by protoplast fusion technique. Xylanase activity in the fusants and parental strains were evaluated and after selection of the best xylanase producer, the expression patterns of the xylanase gene xyn3 was evaluated in the parental strains and the selected fusant using quantitative PCR (qPCR) to figure out validity and success of protoplast fusion.

MATERIALS AND METHODS

Fungal strains and culture media

Sixty Trichoderma isolates were obtained from the Microbial Culture Collection of Agricultural Biotechnology Research Institute of Iran (ABRII). The strains were preserved on potato dextrose agar PDA (Fluka, Germany) at 4 °C. The medium compos-ition for plate enzyme assay was as follows: 4 g/l, K₂HPO₄; 1 g/l, MgSO₄.7H₂O; 0.2 g/l, KCl; 0.02 g/l, FeSO₄·7H₂O; 5 g/l, peptone; 1 g/l, yeast extract; 5 g/l, Oat-spelt xylan; 15 g/l, Agar; pH 7. Protoplast regeneration medium composition was as follows: 1 g/l, yeast extract; 1 g/l, N-Z amine A from bovine milk (Sigma, Germany); 1.6 M sucrose; 16 g/l, Agar. Top agar composition was 15 g/l, KH₂PO₄; 5 g/l, (NH₄)₂SO₄; 20 g/l, Glucose; 182.2 g/l, Sorbitol; 0.3 g/l, MgSO₄.7H₂O; 0.4 g/l, CaCl₂; 0.001 g/l, FeSO₄.7H₂O; 0.0008 g/l, ZnCl₂; 0.0005 g/l, MnSO₄.H₂O; 0.0005 g/l, CoCl₂.6H₂O; 30 g/l, Agar. Mandels and Reese medium (20 g/l, lactose; 1.4 g/l, (NH₄)₂SO₄; 0.3 g/l, urea; 1 g/l, proteose peptone; 2 g/l, KH₂PO₄; 0.3 g/l, MgSO₄.7H₂O; 0.3 g/l, CaCl₂; 0.001 g/l, FeSO₄.7H₂O; 0.0008 g/l, ZnCl₂; 0.0005 g/l, MnSO₄.H₂O; 0.0005 g/l, CoCl₂.6H₂O; pH 4.8) was used for xylanase production assay.

Plate screening for xylanase production

Xylan Agar plates were inoculated with 5 mm diameter mycelial discs from seven days old culture of sixty fungal isolates on PDA, and incubated at 28 °C under a dark condition for three days. Congo Red assay (2.5 g/l) was used to determine the xylanase activity. After 15 minutes of incubation with dye solution, the plates were washed using 1 M NaCl followed by distilled water. The diameter of halos produced by each isolate was measured and used as an indicator of hydrolysis ability (Neethu et al. 2012).

Molecular characterization of selected isolates

Total genomic DNA was extracted from eighteen hyper xylanase producing isolates according to the protocol previously described by Zhong and Steffenson (2001). The quantity of extracted DNAs was analyzed by a nanodrop system (Thermo2000c, USA). PCR amplification was performed for amplification of ITS-rDNA region in 20 µl reaction volume containing 0.2 unit/µl Taq DNA polymerase (Ampliqon, Denmark); 1.5 mM, MgCl₂; 0.2 µM of ITS-1 primer (5'-CGTAGGTGAACCTGCGG-3') and ITS-4 primer (5'-TCCTCCGCTTATTGATATGC-3') and 10 ng genomic DNA of each isolate. PCR amplification was carried out in a PeQSTAR thermocycler (Germany) programmed as follows: an initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 20 s, and extension at 72 °C for 1 min, and the amplification was completed with one cycle of final extension at 72 °C for 5 min. PCR amplicons were resolved by agarose gel electrophoresis and afterward, the obtained sequences (Macrogen, South Korea) were aligned using MEGA 6 and sequence analysis was performed using TrichOKEY 2.0 available online at http://www.isth.info/.

Protoplast isolation

Conidial suspensions of two parental strains (G25-1 and G130-4) were prepared in 0.9% NaCl from seven days fresh plates on PDA. Approximately 10⁶ spores/ml were inoculated on PDA plates covered by cellophane sheets, and 20 plates were prepared for each parental strain. Plates were kept at 28 °C for 16-18 hours. The grown mycelium on cellophane sheets incubated with 50 ml of 1.2 M MgSO₄-10mM Na-phosphate buffer (pH 5.8) containing 10 mg/ml Lysing Enzyme (Sigma, “Gibb.Best”, Germany), 5 mg/ml of Driselase (Fluka, Germany) and 0.1 mg/ml of chitinase from Saccharomyces griseus (Sigma, Germany) and incubated at 28 °C with 100 rpm shaking for 3 hours and the protoplast formation was monitored by hemocytometer. After the incubation, the digested mycelium was filtered through glass filter crucible (pore size 0 and 1) and collected protoplasts were washed with 0.6 M Sorbitol- 0.1 M Tris-HCl pH 7; 1.2 M Sorbitol-10 mM Tris-HCl pH 7.5 and finally the pellets were resuspended in 1 ml of STC buffer (1.2 M of Sorbitol; 10 mM of Tris-HCl pH 7.5, 10 mM of CaCl₂). The amount of protoplast was counted with a hemocytometer.

Protoplast fusion

Protoplast fusion of the parental strains was carried out through Stasz et al., a protocol with minor modifications (Stasz et al., 1988). Equal number of each parental protoplast (10⁶ protoplasts/ml) was mixed with an equal volume of 40% polyethylene glycol (PEG) (MW8000, Sigma, USA) prepared in STC buffer (0.6 M of Sorbitol; 10 mM of Tris-HCl pH 7, 10 mM of CaCl₂), and incubated at 28 °C for 10 min. After adding an equal volume of STC buffer, the tubes were gently inverted and centrifuged for 10 min at 100 rpm. This step was repeated; and finally, the colorless pellet was resuspended in 150 µl of STC buffer. 

Protoplast regeneration

The fusion mixture was incubated on regeneration plates using the top agar method (Penttilä et al. 1987). Regeneration plates were kept at 28 °C, and colony formation was considered. 

Enzyme assay of fusants

Qualitative xylanase assay: The number of seventeen regenerated colonies was transferred to PDA plates and the qualitative assay was carried out using plates containing xylan and 0.1 % Triton™ X-100 (Merck, Germany) and Congo Red method as previously described. Sampling selection was conducted based on halos formation size.

Quantitative xylanase assay: Quantitative xylanase assay was performed by XylX6 assay kit (K-XylX6-2V, Megazyme, Ireland) according to the manufacture procedure. One unit of activity is defined as the amount of enzyme required to release one micromole of 4-nitrophenol from K-XylX6-2V in one minute under the defined assay conditions.

Protein measurement of fusants

The extracellular protein content was estimated using the Bradford method (1976) and bovine serum albumin was used as a standard protein. Additionally, specific enzyme activity was calculated by dividing the number of Units/ml by the protein concentration in mg/ml to get Units/mg.

RNA extraction and quantitative PCR

Total RNA extracted from mycelium of seven days old culture of parental strains G25-1 and G130-4, and fusants in Mandels and Reese medium with TRIzol reagent (Merck, Germany). The quantity and quality of the isolated total RNA were measured by nanodrop and gel electrophoresis. First strand cDNA was synthesized using SuPrimeScript cDNA Synthesis kit (GeNet Bio, Korea) according to the manufacturing procedure. The concentration of 500 ng total RNA with an anchored-oligo (dT)₂₀ and random hexamer primer were applied for cDNA synthesis. Gene expression was monitored by qPCR in 10 µl volume containing 5 ng of cDNA, 5 µl of SYBR green premix (Ampliqon, Denmark) with 50 mM of calibration ROX dye, and 0.2 µM of xyn3 specific primer (xyn3 F 5'-GTACAAGGGCAAGATTCG-3', xyn3 R 5'-AAGGCAATCGAGACAAACT-3') (Rahman et al. 2009). Thermal cycling was performed using StepOnePlus™ Real-Time PCR System (ABI, USA) followed by melting curve analysis with the following cycling program: 15 min at 95 °C, followed by 40 cycles of 15s at 94 °C, the 30s at 58 °C and 20s at 72 °C. The CT data achieved were analyzed by the comparative cycle threshold (CT) method (2^-ΔΔCT) for relative quantification (Livak et al. 2001). The relative expression ratio was normalized by the actin housekeeping gene. All the experiments were conducted in triplicates, and final data analysis was performed using Microsoft Office Excel 2007.

RESULTS AND DISCUSSION

Selection of high xylanase producing isolates

To identify high xylanase producing native Trichoderma isolates, plate screening technique was conducted based on the formation of clear zones by each fungal isolate. According to Ten et al. (2004), the measurement of the halo zone is a useful method for the selection of strains with the efficient capability of polysaccharides degradation. Therefore, a total number of eighteen promising isolates showing maximum clear zones were selected for next in detailed experiments (Fig. 1). Seven isolates, including G104, K2-G139-2, G130-4, G146-5, E(18-10)6, T 7-3 and Mtree1 could provide completely clear plates by degradation of all xylan substrate in the plates (Fig 1). Neethu et al (2012) identified T. viride strains with higher cellulase, hemicellulase and ligninase activity based on plate screening method.

Species identification of the selected isolates

An approximately 400 bp of 5.8S-ITS rDNA fragment was successfully amplified in all eighteen isolates and sequenced for Trichoderma identification (Fig. 2). Sequence analysis of the ITS1-5.8S-ITS2 region is widely used for precise identification of Trichoderma species (Druzhinina et al. 2005). Using bioinformatics tools, two different species, including T. virens and T. harzianum were identified among the selected isolates(Table 1). Parental selection for protoplasting was designed to choose two different species allowing more genetic polymorphism exchange.

Quantitative xylanase assay and selection of parental strains

The quantitative xylanase assay was performed for all the eighteen selected strains by xylanase assay Megazyme kit which measured endo-Xylanase activity. Two strains including T. virens G25-1 and T. harzianum G130-4 with the activity of 0.0568 Uml^-1 and 1.12 Uml^-1, respectively, showed the maximum xylanase activity. These strains were selected as the parents for protoplast fusion, because they showed the highest xylanase activity and moreover, they were two different Trichoderma species so that the creation of genetic combinations could be more accessible. Meanwhile, the lactose 2% was applied as an inducer for xylanase production, since it induces both cellulase and xylanase production pathways (Xiong et al. 2004). XYR1 is a xylanase regulator that regulates lactose metabolism in T. reesei, and subsequently, lactose is able to induce xylanase production (Stricker et al. 2007; da Silva Delabona et al. 2017).

Fig. 1. Congo red assay of the eighteen selected isolates presenting the highest ability to use xylan as a sole carbon source in plate medium after 3 days of incubation at 28°C.

Fig. 2. Amplification of ITS rDNA fragment in the selected Trichoderma isolates. M: 1 kb DNA ladder, T1-T18: eighteen selected Trichoderma isolates

Fig 3. The released protoplasts of the parental Trichoderma strains, a. T. virens G25-1, b. T. harzianum G130-4). 

Fig. 4. a-c. Protoplast fusion monitoring of the parental strains Trichoderma virens G25-1 and  T. harzianum G130-4 by sampling from fusogen solution during 10 minutes incubation of parental protoplasts with PEG at 28 °C.