Enzyme and Microbial Technology 38 (2006) 719–723
Intra-strain protoplast fusion enhances carboxymethyl cellulase activity in Trichoderma reesei V.R. Prabavathy, N. Mathivanan ∗ , E. Sagadevan, K. Murugesan, D. Lalithakumari Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600025, India Received 5 June 2005; received in revised form 30 November 2005; accepted 30 November 2005
Abstract Protoplasts were isolated from Trichoderma reesei strain PTr2 using Lysing enzymes (Sigma Chemicals Co., USA) with 0.6 M KCl as osmotic stabilizer. Intra-strain protoplast fusion has been carried out using 40% polyethylene glycol with STC (sorbitol, Tris–HCl, CaCl2 ) buffer. The fused protoplasts of T. reesei have been regenerated on carboxymethyl cellulose agar (CMCA) selective medium and 15 fusants were selected for further studies. Most of the fusants exhibited fast mycelial growth and abundant sporulation on PDA as compared to non-fusant and parent strains. Prominent clearing zones that appeared around the mycelial growth of most of the fusant progenies when grown on CMCA indicated the secretion of high level extracellular carboxymethyl cellulase (CMCase) than the non-fusants and parent. High CMCase activity was estimated with 80% of the fusants and more than two-fold increase in enzyme activity was recorded with two fusants, SFTr2 and SFTr3 as compared to the parent strain PTr2. Results of the present study demonstrated the scope and significance of the protoplast fusion technique, which can be used to develop superior hybrid strains of filamentous fungi that lack inherent sexual reproduction. © 2005 Elsevier Inc. All rights reserved. Keywords: Trichoderma reesei; Protoplast; Fusion; Regeneration; Carboxymethyl cellulase
1. Introduction Trichoderma reesei is a known producer of cellulolytic enzymes [1] that are extensively used for the degradation and other processes of cellulose materials particularly in textile and paper industries besides, it is also used for wastewater treatment [2]. Fungal protoplasts are an important tool in physiological and genetic research [3,4] and genetic manipulation can successfully be achieved through fusion of protoplasts in filamentous fungi that lack the capacity for sexual reproduction [5]. Hence, protoplast fusion is one of the important approaches in the strain improvement programme [6]. Isolation, fusion and regeneration of protoplasts have been achieved in the genus Trichoderma mainly to enhance its biocontrol potential [7,8]. However, limited attempts were made to improve the strain of Trichoderma to enhance enzyme production [1,9]. Hence, there is ample scope for strain improvement in Trichoderma utilizing this technique
for enhancing the enzyme production. With this background, the present work was aimed to isolate the protoplasts from T. reesei and carry out the intra-strain protoplast fusion with the objective of investigating the possible enhancement of the extracellular carboxymethyl cellulase (CMCase) production in the fusant progenies. 2. Materials and methods 2.1. Culture of T. reesei A large number of Trichoderma strains have been isolated from different soils and decomposed organic matters, characterised, documented and are being maintained at our laboratory. Qualitative and quantitative screening for extracellular lytic enzyme resulted in selecting T. reesei strain PTr2, a producer of CMCase and was used as parent culture for protoplast fusion programme [10]. Stock cultures of T. reesei were maintained on potato dextrose agar (PDA) in test tubes or conical flasks at room temperature (28 ± 2 ◦ C).
2.2. Isolation of protoplasts from T. reesei ∗
Corresponding author. Tel.: +91 44 22350401 (O)/52022789 (R); fax: +91 44 22352494. E-mail address:
[email protected] (N. Mathivanan). 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.11.022
The inoculum of T. reesei (PTr2) was prepared by adding 5 ml of sterile water to 5 days old culture in a conical flask with gentle shaking for 5 min. About 1 ml of inoculum (1 × 107 conidia ml−1 ) from this conidial suspension
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was transferred to 100 ml of potato dextrose broth and incubated for 16 h on a rotary shaker with a speed of 100 rpm at room temperature. The culture was harvested and the young mycelia were separated by filtration using Whatman no. 1 filter paper. About 100 mg fresh mycelium was washed with sterile distilled water followed by 0.1 M phosphate buffer, pH 6.0 and incubated with Lysing enzymes (Sigma Chemicals Co.) at 8 mg ml−1 concentration prepared in phosphate buffer containing 0.6 M KCl as osmotic stabilizer. The mycelia–enzyme mixture was incubated on a shaker with a speed of 75 rpm at room temperature. The lysis of mycelia and the release of protoplasts were monitored at 30 min intervals under light microscope. After 3 h, the enzyme–protoplasts mixture was filtered through a sterile cotton wad and centrifuged at 100 rpm for 10 min. The supernatant was discarded and the protoplast sediment was suspended immediately in buffer–osmotic stabilizer solution.
2.3. Protoplast fusion Intra-strain protoplast fusion in T. reesei was carried out by the method of Stasz et al. [11] with little modification. Polyethylene glycol (PEG) (MW 3500, Sigma Chemicals Co., USA) prepared in STC buffer (0.6 M sorbitol; 10 mM Tris–HCl; 10 mM CaCl2 , pH 6.5) was used as fusogen. One milliliter of protoplast suspension (ca. 1 × 106 protoplasts ml−1 ) was mixed with equal volume of 80% PEG solution and the fusion mixture was incubated at room temperature. After 10 min, the mixture was diluted with 1 ml of STC buffer.
2.4. Regeneration of fused and non-fused protoplasts The PEG in the fusion mixture was washed away twice, using STC buffer and the fused protoplasts of T. reesei were collected by centrifugation at 100 rpm for 10 min. These fused protoplasts were suspended in 100 l of STC buffer and plated on 2% CMCA selective medium. The plates were incubated at room temperature and the regenerated colonies were isolated and subcultured on CMCA and PDA. The fusion protoplasts were also incubated with CMC broth containing 0.6 M KCl as osmotic stabilizer for microscopic observation and photographs were taken using a phase-contrast microscope. Non-fusion protoplasts of T. reesei served as control.
2.5. Growth of fusants, non-fusants and parent of T. reesei on CMCA and PDA About 15 fusants (SFTr1-SFTr15) of T. reesei were selected based on their fast growth on selective medium. Two non-fusants (NFTr1 and NFTr2) have also been selected to compare the variation between fusants, non-fusants and parent. A mycelial disc of each fusant, non-fusant and parent was placed on both CMCA and PDA media in Petri plate in an inverted position and incubated at room temperature. The mycelial growth, morphology, pigmentation and sporulation were observed after 3–5 days.
2.6. Production of CMCase in fusant, non-fusant and parent strains The fusant, non-fusant and parent strains of T. reesei were examined for the production of extracellular CMCase. The CMCB medium consisting of (g l−1 of distilled water) CMC, 5.0; NaNO3 , 2.0; K2 HPO4 , 1.0; KCl, 0.5; MgSO4 , 0.5 and FeSO4 , 0.01, pH 6.5 was used for growing T. reesei. All the strains were grown in 50 ml of CMCB in 250 ml Erlenmeyer flask. Each flask was inoculated with 1 ml conidial suspension (1 × 107 conidia ml−1 ) and incubated at room temperature on a rotary shaker at 100 rpm. Triplicate flasks were maintained for each strain. After 6 days, the cultures were harvested, filtered through Whatman no. 1 filter paper in a glass funnel and the culture filtrates were centrifuged at 10,000 rpm at 4 ◦ C. The cell free culture filtrates were used as enzyme sources for CMCase assay.
2.7. Protein estimation The protein content in the culture filtrates was estimated by the dyebinding method of Bradford [12]. The amount of protein was calculated
using Bovine Serum Albumin fraction V (Sigma Chemicals Co., USA) as standard.
2.8. CMCase assay The assay mixture consisting of 0.1 ml culture filtrate and 0.9 ml suspension of 1% carboxymethyl cellulose (CMC) prepared in 100 mM sodium citrate buffer (pH 5.0) was incubated at 55 ◦ C in a water bath with constant shaking. Controls, without enzyme, substrate and with boiled enzyme were maintained. After 15 min, the reducing sugar formed in the reaction mixture was estimated by the method of Miller [13] using dinitrosalicylic acid reagent. One unit enzyme activity was defined as the amount of enzyme that produced 1 M of reducing sugars ml−1 min−1 from CMC under standard assay conditions using glucose as standard.
3. Results 3.1. Isolation of protoplasts Incubation of T. reesei mycelium with Lysing enzymes (Sigma Chemicals Co.) resulted in lysis of cell wall and release of protoplasts. Swelling and rounding up of cell content were observed initially and subsequently the T. reesei mycelium started lysing after 2 h. Almost complete digestion of mycelia and release of protoplasts occurred prominently after 3 h of incubation (Fig. 1). The protoplasts just released out of mycelium were smaller in size but later they slowly enlarged to a spherical structure. 3.2. Fusion of protoplasts When the protoplasts were mixed with PEG solution, they stuck together and pairs of protoplasts were observed. Later the plasma membranes in the place of contact of both the protoplasts dissolved and fusion of protoplasmic contents took place (Fig. 2). Subsequently the nuclei of the pairing protoplasts fused together (karyogamy) in many cases and in some cases, dikaryotic stage without nuclear fusion was observed. Finally, the fused protoplasts became single, larger and round or oval shaped structures. 3.3. Regeneration of fused and non-fused protoplasts and selection of fusant strains The fused protoplasts of T. reesei started regenerating after 2 days (Fig. 3) and developed mycelium after 3 days on selective medium. The colony development was observed after 4 days on 2% CMCA. Based on the mycelial growth, 15 fast growing colonies of intra-strain fusants of T. reesei were selected and designated as SFTr1-SFTr15. However, the non-fusion protoplasts did not germinate into colonies even after 3 days on selective medium. They normally took more than 4 days to develop into colonies and their growth rates were slow as compared to fusion protoplasts. Two non-fusant regenerated colonies that appeared on 5th day after plating on selective medium were selected based on fast growth as compared to other colonies, which normally required more than 5 days. These were designated as NFTr1 and NFTr2.
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Fig. 1. Isolation of protoplasts in T. reesei. Left: T. reesei mycelium after 90 min of incubation with Lysing enzymes showing swelling and rounding up of cell content (×400). Right: Released protoplasts with few mycelial fragments observed after 3 h of incubation (×400).
ent strains on PDA. Prominent variation in yellow pigmentation among the fusants, non-fusants and parent was observed. Although the growth of fusant, non-fusant and parent strains of T. reesei seems to be apparently similar in CMCA, the clear zone around the mycelium differed distinctly between the strains. Appearance of distinct and larger clearing zones in most of the fusants indicated high levels of extracellular CMCase production in those fusants. Interestingly the fusants strains exhibited much variation in mycelial growth, pigmentation, sporulation and clearing zone formation on CMCA as compared to the parent and non-fusants. However, we observed some variations in the above characteristics between the non-fusants and parent, but these were not significant. Fig. 2. Fusion of protoplasts of T. reesei after treatment with polyethylene glycol (×400).
3.4. Growth of the fusant, non-fusant and parent strains on PDA and CMCA media All the 15 fusants of T. reesei exhibited luxuriant mycelial growth and profuse sporulation than the non-fusant and par-
3.5. Protein content in culture filtrates of the parent, fusants and non-fusants of T. reesei The protein content in culture filtrates of all the 15 fusants ranged between 54 and 78 g ml−1 as against 66 g ml−1 in parent and 68 and 67 g ml−1 in non-fusant strains. Maximum amount of protein was estimated in the fusant SFTr1 and the minimum was in the culture filtrate of the fusants SFTr13 (Fig. 4). 3.6. CMCase activity in culture filtrates of the parent, fusants and non-fusants of T. reesei
Fig. 3. Regeneration of fused protoplasts of T. reesei (×400).
The CMCase activity remarkably increased in most (80%) of the fusants except strains SFTr14, SFTr13 and SFTr15 as compared to the non-fusants (NFTr1 and NFTr2) and parent PTr2. The maximum enzyme activity of 296 unit ml−1 min−1 was estimated in culture filtrate of the fusant SFTr2 and the minimum (92 units ml−1 min−1 ) was recorded in SFTr14. More than two-fold increase in CMCase activity was recorded in 2 out of 15 fusant strains as compared to the parent. Interestingly the fusants SFTr2 registered 2.6-fold increase in CMCase activity than the parent (Fig. 5).
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Fig. 4. Protein content in culture filtrates of the parent, fusants and non-fusants of T. reesei. Values are mean of three replicates with standard deviation.
Fig. 5. CMCase activity in culture filtrates of the parent, fusants and non-fusants of T. reesei. Values are mean of three replicates with standard deviation.
4. Discussion Protoplast fusion is an effective tool for bringing genetic recombination and developing superior hybrid strains in filamentous fungi [6,11,14,15]. In this present study, a CMCase producing T. reesei strain PTr2 was used for intra-strain protoplast fusion programme with the aim of enhancing the extracellular CMCase production. Previously Das et al. [16] demonstrated the intra-strain crossing by protoplast fusion for genetic recombination in Aspergillus niger. Further, Ogawa et al. [1] achieved intra-specific hybridization in T. reesei. The commercial Lysing enzymes (Sigma Chemicals Co.) at 8 mg ml−1 prepared in STC buffer was used to release the protoplasts from T. reesei with 0.6 M KCl as osmotic stabilizer. We have already optimized the conditions for releasing the protoplasts at our laboratory using different permutation combinations in various filamentous fungi including Trichoderma [17]. Interestingly we observed that the release of protoplasts was significantly affected by the concentrations of Lysing enzymes. At low concentrations, the lysis of fungal mycelium took place only at the tip portion resulting in a minimum release of protoplasts whereas at high enzyme concentrations, though the mycelium effectively lysed, the protoplasts bursted immediately after release and disintegrated. Among different concentrations of Lysing enzymes tried, we optimized
that 8 mg ml−1 with 0.6 M KCl as osmotic stabilizer to release higher number of protoplast from different Trichoderma spp. However, Pe’er and Chet [14] obtained highest protoplasts from T. harzianum using Novozym 234 at 10 mg ml−1 with 0.6 M KCl and Tschen and Li [18] used 15 mg ml−1 of Novozym with 0.6 M sucrose to isolate maximum protoplasts from T. harzianum and T. koningii. Further, Balasubramanian et al. [19] obtained maximum number of protoplasts from Trichothecium roseum using Novozym 234 in combination with chitinase and cellulase each at 5 mg ml−1 . Intra-strain protoplasts fusion in T. reesei has been achieved using 40% PEG that was already reported as optimum concentration for inter-specific fusion of protoplasts between T. harzianum and T. longibrachiatum [8]. However, Pe’er and Chet [14] used 33% PEG for intra-specific protoplast fusion in T. harzianum. The concentration of PEG is highly critical for effective fusion of protoplasts. Higher concentrations of PEG caused shrinking and bursting of protoplasts [5,17] and the concentration between 40 and 60% was suitable for protoplasts fusion in different fungi [20,21]. The fusion and non-fusion (control) protoplasts were plated on high concentration (2%) of CMCA for further selection. Though we observed an initial set back in growth of fusants, the colonies exhibited fast mycelial growth after 3 days. However, the non-fusion protoplasts did not germinate into colonies even after 3 days on selective medium. They normally took more than 4 days to develop into colonies and their growth rates were slow as compared to fusion protoplasts. Moreover, the protoplasts, which formed clumps, were not viable and failed to germinate into colonies as already reported [22]. Once subcultured, most of the intra-strain fusants grew very fast on PDA as compared to the non-fusants and the parent, which indicated the quicker adaptability of fusion strains in a newer environment. All the fusant strains grew luxuriantly and sporulated profusely than the parent and non-fusant strains. The intensity of yellow pigmentation in fusant strains was high as compared to non-fusants and parent. Eventhough we could observe some variations between parent and non-fusant strains, these were not significant as already reported from our lab [23]. Although the growth of parent, fusant and non-fusant strains of T. reesei seems to be apparently similar in CMCA, the clear zone around the mycelium differed distinctly between the strains. It was prominent and larger in most of the fusants than the non-fusants and parent indicating the enhanced production of CMCase in those fusants and this could be directly related to strain improvement in Trichoderma. The increased production of CMCase was also confirmed by quantitative assays, in which more than two-fold increase in enzyme activity was recorded with two fusant strains. Although majority of the fusant strains had shown enhanced enzyme activity, few fusants exhibited decreased activity as compared to the parent. This indicated that partial or incomplete genetic recombination might have taken place during protoplast fusion, which could have led to negative effects in some fusants strains. The outcome of the present study has clearly demonstrated the scope and significance of the protoplast fusion technology for developing superior industrially important fungal strains as
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already reported [6]. Further, the intra-strain protoplasts fusion in T. reesei resulted in considerable increase of CMCase activity in most of the fusant strains and more than two-fold increase in enzyme activity in two of the fusant strains has revealed the potential of strain improvement in T. reesei. Hence, this technique can successfully be used to develop superior hybrid strains in filamentous fungi that lack inherent sexual reproduction. Acknowledgements
[10]
[11]
[12]
We thank the Director, CAS in Botany, University of Madras for laboratory facilities. The financial support of Department of Biotechnology, Government of India, New Delhi for this research (Project no. BT\PR\2821\PID\06\144\2001) is gratefully acknowledged.
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