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Induction, screening and identification of Coniothyrium minitans mutants with enhanced β-glucanase activity
Enzyme and Microbial Technology 32 (2003) 224–230
Induction, screening and identification of Coniothyrium minitans mutants with enhanced -glucanase activity J.L. Zantinge a , H.C. Huang b,∗ , K.-J. Cheng c a
Alberta Agriculture (AAFRD), Field Crop Development Centre, 5030 50th Street, Lacombe, Alta., Canada T4L 1W8 b Agriculture and Agri-Food Canada Research Centre, P.O. Box 3000, Lethbridge, Alta., Canada TIJ 4B1 c Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC Received 15 May 2002; received in revised form 9 September 2002; accepted 10 September 2002
Abstract The production of -glucanase in a mycoparasitic fungus, Coniothyrium minitans, was investigated using a wild type strain 2134. Through ultraviolet (UV) irradiation of strain 2134, four mutants, M11-3B2, A7-3D, A8-1 and A10-4, exhibiting enhanced -glucanase activities were isolated. Strains A8-1 and A10-4 were constitutive mutants that expressed barley -glucan hydrolysing activity in the absence of a supplemental inducer (-glucan substrate). Supernatant from A8-1 and A10-4 cultures grown in potato dextrose broth (PDB), the medium without -glucan, had maximum levels of -glucanase activity on average 10 times greater than the wild type strain 2134. M11-3B2 had low levels of constitutive -glucanase expression and enhanced laminarin hydrolysing activity when grown in presence of -glucan-rich substrate. Crown Copyright © 2002 Published by Elsevier Science Inc. All rights reserved. Keywords: Mutants; -Glucans; Coniothyrium minitans
1. Introduction -Glucans are one of the most abundant groups of naturally-occurring polysaccharides. -Glucans with (1,3), (1,4), (1,6) and (1,2) linkages have been identified in both bacteria and plants [1] while (1,3) and (1,6) are abundant in fungal cell walls [2]. Many microorganisms are capable of degrading cellulose and -glucans, by producing enzymes such as cellulases/-1,4-endoglucanase (EC 3.2.1.4) and -glucanases/-1,3(4)-endoglucanses (EC 3.2.1.6.). These enzymes are commercially produced for various industrial applications, by the mass fermentation of high enzyme yielding microorganisms classically generated through mutation, screening and selection. Coniothyrium minitans Campbell is an aerobic mycoparasitic fungus which is capable of attacking sclerotia [3,4] and hyphae [5] of Sclerotinia sclerotiorum (Lib) de Abbreviations: AZCL, Azurine cross-linked; MCD, modified Czapek Dox; MDA, microplate diffusion assay; MRSA, modified reducing sugar assay; PDA, potato dextrose agar; PDB, potato dextrose broth; OBR-HEC, Ostazin Brilliant Red-hydroxyethyl cellulose; UV, ultraviolet ∗ Corresponding author. Tel.: +1-403-317-2226; fax: +1-403-382-3156. E-mail address: [email protected] (H.C. Huang).
Bary. Cell walls of sclerotia and hyphae of S. sclerotiorum are rich in -glucan and chitin [6]. C. minitans produces chitinase and a -glucanase mixture capable of attacking the main carbohydrate component of the cell walls of S. sclerotiorum [7]. C. minitans strain 2134, a wild type strain collected from the Canadian prairies [8], was found to exhibit high levels of -glucanase activity when grown in liquid media or on solid substrates [9]. The strain 2134 also exhibited laminarin hydrolysing activity, and lower levels of carboxymethylcellulose hydrolysing activity (Zantinge et al., unpublished data) as well as antibiotic activity associated with the production of benzofuranones and chromanes [10]. Prior to this study, very little information was available on -glucanase expression or mutation in C. minitans. Recently, the exo--1,3-glucanase (EC 3.2.1.58) was cloned from C. minitans [11] but there is still little information about C. minitans or other mycoparasitic fungal endo-1,3:1,4--glucanases. The objective of our study, to induce and select new mutants from the C. minitans wild type strain 2134 with enhanced and constitutive -glucanase activity using UV irradiation. This is the first report of the production of C. minitans mutants with enhanced and/or constitutive -glucanase expression.
0141-0229/02/$ – see front matter. Crown Copyright © 2002 Published by Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 4 9 - 1
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2. Materials and methods
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0.2% barley--glucan, or 0.2% “inducer” as the only carbohydrate source.
2.1. Fungal strains The wild type of C. minitans strain 2134, was collected from the Canadian prairies [8]. The mutant strains M11-3B2, A10-4, and A8-1 were derived from the strain 2134 after UV irradiation. All stock cultures were stored in 10% glycerol under liquid nitrogen at the Lethbridge Research Centre type culture collection. Working cultures were maintained on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) for up to 4 weeks. 2.2. Mutagenesis and isolation of mutants 2.2.1. Method A: ultraviolet (UV) irradiation of pycnidiospores on agar media Pycnidiospores of C. minitans strain 2134 were streaked onto PDA in Petri dishes. After a 48 h incubation at 20 ◦ C, individual germinated spores were aseptically transferred onto 0.2% Azurine cross-linked (AZCL)--glucan modified Czapek Dox (MCD) agar (l−1 : NH4 H2 PO4 , 2.0 g; K2 HPO4 , 1.0 g; MgSO4 ·7H2 O, 0.5 g; KCl, 0.5 g; FeSO4 , 0.01 g; 1% ZnSO4 , 1 ml; 0.5% CuSO4 , 1 ml; Agar, 20.0 g; AZCL--glucan, 2.0 g (MegaZyme, Australia)). The germinated spore isolates were then exposed to short wave (254 nm) UV germicidal lamp (model 782 L30, Westinghouse Electric Company, Pittsburgh, PA, USA) at a distance of 30 cm for 0 (control plate), 1, 2.5, or 5 min. Plates were placed at 20 ◦ C, in the dark for the first 48 h to prevent photoreactivation and then transferred to light for an additional 5 days. The -glucanase activity was directly monitored on the 0.2% AZCL--glucan MCD agar medium by measuring the clearing zones/blue halos surrounding each colony. Under a dissecting microscope, single hyphal tip isolations were made aseptically from the colonies showing large clearing zones and transferred onto PDA and 0.2% AZCL--glucan MCD agar. Cultures were grown in full light (12.5 einstein m−2 s−1 ) at 20 ◦ C. Phenotypic changes in colony colour, culture shape, culture size and sporulation were recorded at 14 days post inoculation. In addition, mycelia plugs were removed from the edge of duplicate mutant cultures grown on PDA using cork borer #3 (6 mm in diameter) after 4–5 days incubation at 20 ◦ C under light. Three plugs were placed in 20 ml of MCD broth + 1% “inducer” (ground powder of sclerotia of S. sclerotiorum which is rich in glucan [12]) in a 125-ml Erlenmeyer flask. Cultures were incubated on a shaker at 200 rpm, at 20 ◦ C under continuous light (12.5 einstein m−2 s−1 ) and assayed for extracellular enzyme activity at 3 and 5 days by the modified reducing sugar assay (MRSA, described under Section 2.4). In addition, cultures were also tested on potato dextrose broth (PDB) + 1% “inducer” in the same manner described above. High enzyme-producing cultures were repeatedly sub-cultured on MCD broth supplemented with 0.2% AZCL--glucan,
2.3. Method B: UV irradiation of pycnidiospores in suspension Pycnidiospores of C. minitans strain 2134 were harvested from the surface of 14-day-old PDA cultures and suspended in 0.2% barley -glucan MCD broth (l−1 : NH4 H2 PO4 , 2.0 g; K2 HPO4 , 1.0 g; MgSO4 ·7H2 O, 0.5 g; KCl, 0.5 g; FeSO4 , 0.01 g; 1% ZnSO4 , 1 ml; 0.5% CuSO4 , 1 ml; and barley -glucan, 2.0 g (MegaZyme, Australia)) to a final concentration 1×104 pycnidiospores ml−1 . A 10 ml volume of suspension was placed in an open Petri dish (8.7 mm diameter) and exposed to a short wave UV light germicidal lamp (model 782 L30, Westinghouse Electric Company, Pittsburgh, PA, USA) at a distance of 10 cm. A 1 ml control sample was removed from the plate prior to UV exposure. To identify the optimal length of UV exposure, samples were removed after 1, 3, and 5 min. The length of exposure to UV was then adjusted to 3 min for all subsequent experiments as this time was found to kill 97–99% of the pycnidiospores. After irradiation 200 l of spore suspension was transferred into each well of a microplate (or 1 × 103 spores per well and approximately 10–30 viable spores per well). Plate column 1 was used as a control lane, which contained 200 l of non-irradiated spores. Plates were placed at 20 ◦ C in the dark for the first 48 h to prevent photoreactivation and then transferred to full light at 20 ◦ C for an additional 14 days. To select constitutive mutants for -glucanase activity, pycnidiospores were UV irradiated in an identical method as described above, except the suspensions were prepared in PDB. A 50 ml sample of spent culture supernatent taken from each well of the culture plate, was screened by the microplate diffusion assay (MDA) for high -glucanase activity as previously described [9,13]. The commercially available -1,4-endoglucanase (EC 3.2.1.4, Megazyme) at concentrations of 2, 1, 0.5, 0.25, 0.125, 0.06 and 0.00 U ml−1 (diluted in 25 mM Na acetate, pH 4.5) were used as standards. The amount of mycelia present per well of the culture plate was estimated by reading the optical density at a wavelength of 650 nm [14]. The wells with relatively high enzyme levels were transferred onto 0.2% AZCL--glucan agar plates. After 3–4 days individual hyphal tips (10/plate) were isolated under a dissecting microscope from the colony, transferred onto PDA in Petri dishes, and incubated at 20 ◦ C under light for 4–5 days. Single hyphal tip cultures that differed phenotypically, were selected and tested for enzyme production. Mycelia plugs were removed from the edge of the mutant cultures on PDA using cork borer #3, and placed in 3 ml of MCD broth + 1% “inducer” medium in 15 ml test tubes with three plugs per tube. The test tubes were placed on an angle in a test tube rack on a shaker, and incubated at 20 ◦ C, in full light while shaken at 200 rpm. At 7 and 14 days, cultures were tested for enzyme activity using the MDA [9,13] described above. The highest -glucanase-producing
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C. minitans strains were stored in liquid nitrogen, and single hyphal tips were sub-cultured, transferred to PDA/0.2% AZCL--glucan MCD agar and transferred to PDA to allow phenotypic characterization (described below). To confirm the -glucanase production, cultures were again grown in 3 ml of MCD broth + 1% “inducer” and again retested using the MDA. The highest enzyme producing cultures were then grown in 10 ml of MCD broth + 1% “inducer” per 50-ml flask, for 7–14 days and tested for -1,4:1,3-glucanase activity using the MRSA (described below). Constitutive mutants were screened on PDB. Enzyme production of mutant strains was compared to the enzyme activity of the parental strain 2134. 2.4. Characterization of mutants 2.4.1. Enzyme production Extracellular enzyme production was measured from C. minitans wild type strain 2134 and derived mutants (M11-3B2, A10-4, and A8-1). They were grown in PDB, PDB + 1% “inducer”, and MCD broth + 1% “inducer”. Three mycelia plugs from each strain, were removed using cork borer #3 from the edge of 5-day-old colonies on PDA, and used to inoculate 50 ml of broth in a 250-ml Erlenmeyer flask. Fungal cultures were grown in shake cultures (200 rpm) at 20 ◦ C under continuous light (12.5 einstein m−2 s−1 ). Cultures were sampled at 3, 6, 9, 12, and 15 days after inoculation by aseptically removing 1 ml of growth media from each flask. Samples were collected in a 1.5 ml Eppendorf tubes and centrifuged at 5000 × g for 5 min. Extracellular enzyme activities of C. minitans cultures grown in PDB were measured in sample supernatant by the MDA [9,13] and Ostazin Brilliant Red-hydroxyethyl cellulose (OBR-HEC, Sigma #O-6879) [15]. The MDA was used to screen for high -1,4-glucanase (EC 3.2.1.4) utilized 0.2% AZCL-HE-cellulose (Megazyme). Assays were performed in duplicate. Cultures grown on MCD media were assayed using the MRSA. Assays were performed in a 96-well titration plate [16]. Fifty microliters of sample supernatant/enzyme was added to each well and equilibrated to 37 ◦ C. Then, 50 l of carboxmethylcellulose (#C-5013 Sigma–Aldrich Corp., St. Louis, MO, USA), lichenan (Sigma #L-6133), laminarin (Sigma #L-9634) or -glucan (Megazyme-P-BGBH) diluted in 25 mM sodium acetate, pH 4.5, at a concentration of 5 mg ml−1 and equilibrated to 37 ◦ C was added to the enzyme. Assays were allowed to react for 15 min at 37 ◦ C and were stopped by the addition of 100 l of cold solution B (0.3% 3,6-dinitrophthalic acid) and solution C (1.8 M K2 CO3 , +0.1 M Na2 S2 O3 ) mixed prior to use (1:1 v/v). Samples were heated for 45 min at 95 ◦ C, until colour change was completed. The absorpbance was measured at 410 nm using a plate reader (SLT instruments, 16895, Tecan US Inc., Durham, NC, USA). Background absorpbance was measured by a control sample subtracted from the sample
absorpbance. Control reactions were prepared for each sample, following the same method outlined above for the test assays without incubation. One unit of enzyme activity was defined as the amount of enzyme required to release reducing sugars equivalent to 1 mol of glucose per min per ml at 37 ◦ C. 2.4.2. Dry matter weight The solid mass formed in the fungal cultures (45 ml after sampling) were collected at 15 days after growing on PDB, PDB + 1% “inducer”, and MCD broth + 1% “inducer” and centrifuged at 5000 × g for 10 min. Mycelia pellets were transferred into aluminium foil measuring dishes and heated for 48 h at 90 ◦ C. The remaining dry mass in each plate was weighed. 2.4.3. Phenotypic characterization To characterize the C. minitans parental and mutant cultures, a cork borer #3 was used to remove agar plugs containing mycelial mats from the edge of 4–5 days old, PDA cultures. The mycelia plugs were placed on PDA, 0.2% AZCL--glucan supplemented PDA (for constitutive mutants), 0.2% barley -glucan MCD agar, or 0.2% AZCL--glucan MCD agar plates with one plug at the centre of each Petri dish. Cultures were grown in full light (12.5 einstein m−2 s−1 ) at 20 ◦ C. Phenotypic changes in colony colour, culture shape, culture size and sporulation were recorded at 14 days post inoculation.
3. Results and discussion 3.1. Method A: UV irradiation of pycnidiospores on agar The results obtained from method A demonstrated the feasibility of selection of C. minitans strains for enhanced -glucanase production by UV irradiation. UV irradiation was used in this study because of its simplicity, however, the use of other mutagens such as nitrosoguanidine (NTG) may improve the rate of mutant production. Approximately, 50 colonies of C. minitans mutants were obtained from the wild type strain 2134 through UV irradiation. The survival rate of pycnidiospores of C. minitans was approximately 88, 67 and 50% after UV irradiation for 1, 2.5 and 5 min, respectively. Approximately, 50% of the single spore cultures that survived UV irradiation produced clearing zones of greater than 3 mm after 7 days on 0.2% AZCL--glucan MCD agar. These cultures were further purified by single hyphal tip isolations onto PDA and 0.2% AZCL--glucan MCD agar. The primary mutant culture, M11 produced by 1 min exposure to UV irradiation, was determined to have relatively high -glucanase production when screened on 0.2% AZCL--glucan MCD agar. Culture M11-3 (1.88 U ml−1 ), a single spore culture derived from original M11 mutant was about five times higher for -glucanase expression than the
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wild type strain 2134 (0.33 U ml−1 ) after 5 days in MCD broth + 1% “inducer”. In an attempt to further increase -glucanase production by M11-3, it was continuously sub-cultured on 0.2% AZCL--glucan MCD agar. Mutant M11-3B2 was a descendant of the original mutant M11 following eight passages on 0.2% AZCL--glucan MCD agar. Difference in morphological characteristics between mutant M11-3B2 and wild type strain 2134, were evident in cultures grown on PDA for 14 days at 20 ◦ C under light. Colonies of C. minitans wild type strain 2134 have golden-brown pigmentation, smooth edges, and are spore-producing. Colonies of C. minitans mutant strain M11-3B2 have olivaceous golden-brown pigmentation, smooth edges and are spore forming and grew at a faster rate than 2134. Mutant strains derived from the original mutant M11 were unstable when grown on PDA for extended periods, resulting in a decline in enzyme activity. Therefore, passage on a minimal medium such as 0.2% AZCL--glucan MCD agar was essential for culture maintenance.
and screened for -glucanase expression by the MDA. Constitutively expressing -glucanase cultures were identified by measuring -glucanase activity in PDB. Strains A8-1 and A10-4 were identified as constitutive -glucanase producing mutants. Colonies of strain A8-1 grown on PDA at 20 ◦ C under light have yellowish-white pigmentation, serrated edges, and produce very few spores where as colonies of A10-4 have yellowish white pigmentation, serrated edges and produce very few spores. Wild type strain 2134, when cultured on PDA supplemented with 0.2% AZCL--glucan, did not produce a visible zone of clearing surrounding the colony (Fig. 1). Strain 2134, will not produce -glucanase until after the starches and simple sugars have been utilized. The mutant strains, A10-4 and A8-1 with constitutive expression have a visible zone of clearing surrounding the fungal colony. Measuring from outer mycelia edge of the colony, their clearing zones were approximately 5–7 mm wide. Mutant M11-3B2 had a smaller zone of clearing (approximately 3 mm wide).
3.2. Method B: UV irradiation of pycnidiospores in suspension
3.3. Fungal growth and enyzme production
Method B increased the speed of mutant screening and selection. Approximately, 6000 viable UV irradiated pycnidiospores of C. minitans wild type strain 2134 were screened using 0.2% -glucan MCD broth as the initial growth medium. In this approach, mutated spore cultures having a mutation up-regulating -glucanase activity, should grow faster and out compete mycelia from spores with a mutation down-regulating -glucanase activity or no mutation for -glucanase activity. Using the single hyphal tip isolation method, 75 mutant strains were obtained from the UV-treated cultures, which showed relatively high -glucanase activities in the screening tests of microplate wells. These mutants were twice tested for -glucanase production by growing in 3 ml of MCD broth + 1% “inducer”. Of these mutants, 26 mutants were re-tested a third time for enzyme production by growing cultures in a larger 10 ml volume of MCD broth + 1% “inducer” in 50-ml Erlenmeyer flask. The highest enzyme-producing mutant was the mutant strain A7-3D. Colonies of strain A7-3D grown on PDA at 20 ◦ C under light for 14 days have golden-brown pigmentation, serrated edges and produce spores. Approximately, 4000 UV irradiated viable pycnidiospores of C. minitans wild type strain 2134 were grown in PDB and screened for -glucanase activity. We selected for constitutive mutants by using PDB as the selection medium, as non-constitutive fungal isolates would be repressed by simple sugars present in the PDB. Of the 352 wells screened two wells gave relatively high -glucanase activity with relatively low amounts of mycelia present in the well. The mycelia from these wells were transferred and grown on PDA. Single hyphal tip isolations were made from the colony to establish pure cultures on PDA. The cultures derived from these two wells were grown on 3 ml PDB test tube cultures
The average dry mycelial weight showed that each of the four strains of C. minitans grew the best in PDB + 1% “inducer”, followed by PDB and then MCD broth + 1% “inducer” (Table 1). PDB is rich in nutrients (starches and simple sugars). The 1% “inducer” is a natural source of carbohydrate, primarily consisting of -glucan [12]. As expected, the cultures grew best in PDB + 1% “inducer” due to the greater amount simple carbohydrate present. The wild type strain 2134 grew faster in PDB than the mutant strains, which therefore correlated with the larger colony size of strain 2134 on PDA. The characterization of enzyme expression by the wild type strain 2134 and its mutants M11-3B2, A10-4, and A8-1 in various media demonstrated the importance of induction and repression in the control of -glucanase expression. The barley -glucan and cellulose hydrolyzing activity in wild type strain 2134 was greatly repressed by glucose and simple sugars when grown in PDB, producing insignificant amounts of extracellular -glucanase and cellulase. While, mutants A10-4 and A8-1, both selected on PDB, were not repressed by the presence of glucose in PDB and mutant M11-3B2 was only weakly repressed. Mutant A8-1 produced an average 2.74 ± 0.55 U ml−1 of barley -glucan hydrolyzing activity in PDB after 15 days and mutant A10-4 produced an average 2.21 ± 0.34 U ml−1 . Similarly, mutants A10-4 and A8-1 produce extracellular cellulose hydrolyzing activity, assayed at 0.90 ± 0.07 U ml−1 by 6 days and 0.91 ± 0.12 U ml−1 by 9 days, respectively. Mutant M11-3B2 produced lower levels at 0.22 ± 0.03 U ml−1 cellulose hydrolysing activity by 9 days. Wild type strain 2134 produced its highest amounts of enzyme activity when grown on MCD broth supplemented only with “inducer” (Table 2). The “inducer” (ground sclerotia of S. sclerotiorum) enhanced -glucanase expression
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Fig. 1. C. minitans wild type strain 2134 and its mutant strains grown under continuous light at 20 ◦ C for 14 days on PDA supplemented with 0.2% AZCL--glucan. Clearing zones around fungal colonies demonstrate constitutive -glucanase activity. Clearing zones were measured from the outer mycelia colony edge to the outer edge of the clearing. Top row from left to right: 2134, A10-4 and A8-1. Bottom row from left to right: A7-3D, M11-3 and M11-3B2.
in the wild type strain 2134 and mutant M11-3B2 when supplemented into media such as MCD or PDB that is low in -glucan content. When strain 2134 was grown in PDB with 1% “inducer” it began to produce cellulase after a 6–9-day delay, exhibiting approximately 0.22 ± 0.01 U ml−1 by 12 days. When, strain 2134 was grown in MCD broth + 1% “inducer” by 15 days it produced 3.35 ± 0.40 U ml−1 of cellulose hydrolyzing activity while the mutant M11-3B2 only had 2.38 ± 0.16 U ml−1 of cellulose hydrolyzing activity. These results substantiate reports that the expression of genes encoding cell wall degrading enzymes from mycoparasitic fungi, is enhanced by the addition of autoclaved mycelium, fungal cell wall extracts, or polymers, such as laminarin and chitin [11,17,18]. It is generally thought that small diffusible molecules derived from host cell walls trigger greater enzyme expression. Interestingly, the “inducer” did not enhance cellulase expression in constitutive mutants, A10-4 and A8-1, suggesting genes and/or regulatory elements associated with cellulase induction were also mu-
tated. Physiological stress and carbon starvation may also be involved in the up regulation of some of the genes encoding fungal cell wall degrading enzymes [18–20]. Mutant M11-3B2, which was weakly repressed, utilized the simple sugars present in the PDB media quicker than the wild type strain 2134 and cellulase expression was greatly enhanced by the presence of “inducer”. Wild type stain 2134 required “inducer” to weakly overcome the glucose and simple sugar repression. The results of our study, suggest a multiple regulatory mechanisms involved in cellulase and -glucanase expression and that repression and induction mechanisms act independently of each other. Only the cultures grown in MCD broth + 1% “inducer” could be analysed by the MRSA, as cultures grown in PDB based media have high reducing sugar levels that result in high background values. The results of the MRSA are shown in Table 2, using laminarin (a) barley -glucan (b) lichenan (c) and CMC (d) as substrates. The CMC substrate is made of only -1,4-d-glucan. The laminarin substrate is
Table 1 Comparison of dry mycelia weighta of C. minitans: wild strain 2134 and its mutant strains A8-1, A10-4, and M11-3B2, grown in liquid mediab Growth medium
PDB PDB + I MCD + I a b
Dry mycelia weight (g per culture) 2134
A8-1
A10-4
M11-3B2
0.61 ± 0.16 0.65 ± 0.12 0.42 ± 0.13
0.43 ± 0.04 0.70 ± 0.06 0.46 ± 0.07
0.45 ± 0.03 0.80 ± 0.03 0.48 ± 0.03
0.54 ± 0.06 0.87 ± 0.25 0.50 ± 0.014
Average dry mycelial weight from 45 ml cultures. PDB (potato dextrose broth), PDB + I (potato dextrose broth + 1% “inducer”) or MCD + I (MCD broth + 1% “inducer”).
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Table 2 Comparison of production of extracellular -glucanases by C. minitans parental strain 2134 and its mutant strains, A8-1, A10-4 and M11-3B2 after 15 days post inoculation Strains
Enzyme activity (U ml−1 ) Cellulasea PDB + Ie
A8-1 A10-4 M11-3B2 2134
0.29 0.16 2.04 0.21
± ± ± ±
0.01 0.05 0.05 0.01
MCD + Ie 0.04 0.91 2.38 3.35
± ± ± ±
0.02 0.11 0.16 0.40
Laminaraseb
-Glucanasec
Lichenased
MCD + Ie
MCD + Ie
MCD + Ie
5.33 4.33 7.22 5.99
± ± ± ±
0.14 1.1 0.49 0.29
5.48 6.51 6.50 6.00
± ± ± ±
0.29 0.35 0.35 0.04
4.86 4.26 6.51 4.46
± ± ± ±
0.42 0.37 0.15 0.21
a Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: cellulose for cellulase activity (-1,4-endoglucanase activity). b Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: laminarin for laminarinase activity (-1,3-endoglucanase activity). c Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: barley -glucan for barley -glucanase activity (-1,3 and 1,4-endoglucanase activity). d Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: lichenan for lichenase activity (-1,3 and 1,4-endoglucanase activity). e Culture media: C. minitans strains were grown in either PDB + 1% “inducer” (PDB + I), or MCD broth + 1% “inducer” (MCD + I). Cellulose hydrolyzing activity is measured by the OBR-HEC assay method for PDB + I, and by the MRSA for MCD + I.
made of only -1,3-d-glucan. Lichenan and barley -glucan consists of both 1,3 and 1,4 -d-glucan. Wild type strain 2134 produced the highest level of cellulose hydrolyzing activity (3.35 ± 0.40 U ml−1 ) while the mutant M11-3B2 produced the highest level of laminarin hydrolyzing activity (7.22 ± 0.49 U ml−1 ) by 15 days. Also, M11-3B2 mutant strain produced more lichenan hydrolyzing activity (6.50 ± 0.35 U ml−1 ) and similar amounts of barley -glucan hydrolyzing activity (6.51 ± 0.15 U ml−1 ) as the wild type strain 2134 (4.46 ± 0.21 and 6.00 ± 0.04 U ml−1 , respectively). The substrate lichenan has a higher ratio of -1,3 to -1,4 linkages than barley -glucan, thereby explaining the slightly enhanced barley -glucan hydrolyzing activity of strain 2134. Mutants A8-1 and A10-4 produced very low levels of cellulase, but still produced 4.33±1.1 and 5.33 ± 0.14 U ml−1 , respectively of laminarin hydrolyzing activity which was only slightly lower than strain 2134. Various applications have been suggested for the utilisation of cell wall degrading -glucanase activity, such as their use in agriculture as feed additives [21] or their production by superior strains of C. minitans as biocontrol agents [22]. A mutated C. minitans strain with superior -glucanase activity could ultimately be used to produce -glucanase on an inexpensive crude substrate such as a food processing waste product. For large scale economic fermentation of C. minitans strains having -glucanase activity, it is desirable to have constitutive -glucanase activity that does not require induction by a source of -glucan and which is not repressed by the presence of simple sugars. Although natural sources of -glucan are available, such as the “inducer” obtained from the sclerotia of S. sclerotiorum, many inexpensive and readily available growth media, such as potato skins, do not provide a -glucan source. It is preferable not to have to add a -glucan source to the culture media. The creation of stable C. minitans mutants with enhanced
and/or constitutive -glucanase expression is the first step in: developing effective mutagenesis, screening and selection methods; increasing our knowledge and understanding of constitutive expression; maximising -glucanase expression; and developing good candidate fungal strains for use in potential commercial enzyme fermentation systems.
4. Conclusions From this study, it is concluded that UV irradiation can induce C. minitans mutants that are culturally stable. The control of -glucanase expression is multigenic and intricate. Selective screening methods such as initial growth on PDB or 0.2% barley -glucan MCD broth followed by the MDA can be applied successfully to isolate specific mutants with constitutive and/or enhanced -glucanase expression.
Acknowledgments The authors would like to thank J. Yanke for maintaining the stock cultures of C. minitans for this study at the Culture Collection Centre, Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alta., Canada. References [1] Stone BA, Clarke AE. Chemistry and biology of (1–3)--glucans. Melbourne, Australia: La trobe University Press; 1992. [2] Wessels JGH, Seitsma JH. Fungal cell walls: a survey. In: Tanner W, Loewus FA, editors. Encyclopedia of plant physiology 13B. Berlin: Springer; 1981. p. 352–94. [3] Huang HC, Hoes JA. Penetration and infection of Sclerotinia sclerotiorum by Coniothyrium minitans. Can J Bot 1976;54:406–10.
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[4] Huang HC, Kokko EG. Ultrastructure of hyperparasitism of Coniothyrium minitans on sclerotia of Sclerotinia sclerotiorum. Can J Bot 1987;65:2483–9. [5] Huang HC, Kokko EG. Penetration of hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans without the formation of appressoria. Phytopathol Zeit 1988;123(1):133–9. [6] Jones D. Ultrastructure and composition of the cell walls of Sclerotinia sclerotium. Trans Br Mycol Soc 1970;54:351–60. [7] Jones D, Gordon A, Bacon J. Co-operative action by endo and exo--(1–3)-glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotium (Lib) de bary. Biochem J 1974;140:47–55. [8] Huang HC. Distribution of Coniothyrium minitans in Manitoba sunflower fields. Can J Plant Pathol 1981;3:219–22. [9] Huang HC, Cheng K-J, Zantinge JL, Laroche A. Strains of Coniothyrium minitans having -glucanase activity. US Patent no. 6,268,202 B1 (2001). [10] Machida K, Trifonov LS, Ayer WA, Lu Z-H, Laroche A, Huang HC, et al. 3(2H)-Benzofuranones and chromanes from liquid cultures of the mycoparasitic fungus Coniothyrium minitans. Phytochemistry 2001;58:173–7. [11] Giczey G, Kerenyl Z, Fulop L, Hornok L. Expression cmg1, an Exo-1,3-glucanase gene from Coniothyrium minitans, increases during sclerotial parasitism. Appl Environ Microbiol 2001;67:865–71. [12] Saito I. Studies on the maturation and germination of sclerotia of Sclerotinia sclerotiroum (lib) de Bary, a casual fungus of bean stem rot. Hokkaido Prefectural Agric Exp Sta Bull 1977;26:106. [13] Zantinge JL, Huang HC, Cheng K-J. Microplate format diffusion assay for rapid screening of -glucanase producing microorganisms. Biotechniques 2002;33:798–806.
[14] Mohler WA, Charlton CA, Blau HM. Spectrophotometric quantitation of tissue culture cell number in any medium. Biotechniques 1996;21:260–6. [15] Biely P, Mislovicova D, Toman R. Soluble chromogenic substrates for the assay of endo-1,4--glucanase. Anal Biochem 1985;144:142–6. [16] Zheng Y, Wozniak CA. Adoption of a -1,3-glucanase assay to microplate format. Biotechniques 1997;22:922–6. [17] Cohen-Kupiec R, Broglie KE, Friesem D, Broglie RM, Chet I. Molecular characterization of a novel -1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum. Gene 1999;226: 147–54. [18] Mach RL, Peterbauer CK, Payr K, Jaksits S, Woo SL, Zeilinger S, et al. Expression of two major chitinase genes of Trichoderma atroviride (harzianum P1) is triggered by different regulatory signals. Appl Environ Microbiol 1999;65:1858–63. [19] Lora JM, de la Cruz J, Llobell A, Benetez T, Pinto-Toro JA. Molecular characterization and heterologous expression of an endo-beta-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum. Mol Gen Genet 1995;247:639–45. [20] Margolles-Clark EL, Harman GE, Penttila ME. Enhanced expression of endochitinase in Trichoderma harzianum with the cbhl promoter of Trichoderma reesei. Appl Environ Microbiol 1996;62:2152–5. [21] Ouhida I, Perez JF, Gasa J, Puchal F. Enzymes (beta-glucanase and arabinoxylanase) and/or sepiolite supplementation and the nutritive value of maize–barley–wheat based diets for broiler chickens. Br Poult Sci 2000;41(5):617–24. [22] de Vrije T, Antoine N, Buitelaar RM, Bruckner S, Dissevelt M, Durand A, et al. The fungal biocontrol agent Coniothyrium minitans: production by solid-state fermentation. Appl Microbiol Biotechnol 2001;56:58–68.
Induction, screening and identification of Coniothyrium minitans mutants with enhanced -glucanase activity J.L. Zantinge a , H.C. Huang b,∗ , K.-J. Cheng c a
Alberta Agriculture (AAFRD), Field Crop Development Centre, 5030 50th Street, Lacombe, Alta., Canada T4L 1W8 b Agriculture and Agri-Food Canada Research Centre, P.O. Box 3000, Lethbridge, Alta., Canada TIJ 4B1 c Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC Received 15 May 2002; received in revised form 9 September 2002; accepted 10 September 2002
Abstract The production of -glucanase in a mycoparasitic fungus, Coniothyrium minitans, was investigated using a wild type strain 2134. Through ultraviolet (UV) irradiation of strain 2134, four mutants, M11-3B2, A7-3D, A8-1 and A10-4, exhibiting enhanced -glucanase activities were isolated. Strains A8-1 and A10-4 were constitutive mutants that expressed barley -glucan hydrolysing activity in the absence of a supplemental inducer (-glucan substrate). Supernatant from A8-1 and A10-4 cultures grown in potato dextrose broth (PDB), the medium without -glucan, had maximum levels of -glucanase activity on average 10 times greater than the wild type strain 2134. M11-3B2 had low levels of constitutive -glucanase expression and enhanced laminarin hydrolysing activity when grown in presence of -glucan-rich substrate. Crown Copyright © 2002 Published by Elsevier Science Inc. All rights reserved. Keywords: Mutants; -Glucans; Coniothyrium minitans
1. Introduction -Glucans are one of the most abundant groups of naturally-occurring polysaccharides. -Glucans with (1,3), (1,4), (1,6) and (1,2) linkages have been identified in both bacteria and plants [1] while (1,3) and (1,6) are abundant in fungal cell walls [2]. Many microorganisms are capable of degrading cellulose and -glucans, by producing enzymes such as cellulases/-1,4-endoglucanase (EC 3.2.1.4) and -glucanases/-1,3(4)-endoglucanses (EC 3.2.1.6.). These enzymes are commercially produced for various industrial applications, by the mass fermentation of high enzyme yielding microorganisms classically generated through mutation, screening and selection. Coniothyrium minitans Campbell is an aerobic mycoparasitic fungus which is capable of attacking sclerotia [3,4] and hyphae [5] of Sclerotinia sclerotiorum (Lib) de Abbreviations: AZCL, Azurine cross-linked; MCD, modified Czapek Dox; MDA, microplate diffusion assay; MRSA, modified reducing sugar assay; PDA, potato dextrose agar; PDB, potato dextrose broth; OBR-HEC, Ostazin Brilliant Red-hydroxyethyl cellulose; UV, ultraviolet ∗ Corresponding author. Tel.: +1-403-317-2226; fax: +1-403-382-3156. E-mail address: [email protected] (H.C. Huang).
Bary. Cell walls of sclerotia and hyphae of S. sclerotiorum are rich in -glucan and chitin [6]. C. minitans produces chitinase and a -glucanase mixture capable of attacking the main carbohydrate component of the cell walls of S. sclerotiorum [7]. C. minitans strain 2134, a wild type strain collected from the Canadian prairies [8], was found to exhibit high levels of -glucanase activity when grown in liquid media or on solid substrates [9]. The strain 2134 also exhibited laminarin hydrolysing activity, and lower levels of carboxymethylcellulose hydrolysing activity (Zantinge et al., unpublished data) as well as antibiotic activity associated with the production of benzofuranones and chromanes [10]. Prior to this study, very little information was available on -glucanase expression or mutation in C. minitans. Recently, the exo--1,3-glucanase (EC 3.2.1.58) was cloned from C. minitans [11] but there is still little information about C. minitans or other mycoparasitic fungal endo-1,3:1,4--glucanases. The objective of our study, to induce and select new mutants from the C. minitans wild type strain 2134 with enhanced and constitutive -glucanase activity using UV irradiation. This is the first report of the production of C. minitans mutants with enhanced and/or constitutive -glucanase expression.
0141-0229/02/$ – see front matter. Crown Copyright © 2002 Published by Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 4 9 - 1
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2. Materials and methods
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0.2% barley--glucan, or 0.2% “inducer” as the only carbohydrate source.
2.1. Fungal strains The wild type of C. minitans strain 2134, was collected from the Canadian prairies [8]. The mutant strains M11-3B2, A10-4, and A8-1 were derived from the strain 2134 after UV irradiation. All stock cultures were stored in 10% glycerol under liquid nitrogen at the Lethbridge Research Centre type culture collection. Working cultures were maintained on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) for up to 4 weeks. 2.2. Mutagenesis and isolation of mutants 2.2.1. Method A: ultraviolet (UV) irradiation of pycnidiospores on agar media Pycnidiospores of C. minitans strain 2134 were streaked onto PDA in Petri dishes. After a 48 h incubation at 20 ◦ C, individual germinated spores were aseptically transferred onto 0.2% Azurine cross-linked (AZCL)--glucan modified Czapek Dox (MCD) agar (l−1 : NH4 H2 PO4 , 2.0 g; K2 HPO4 , 1.0 g; MgSO4 ·7H2 O, 0.5 g; KCl, 0.5 g; FeSO4 , 0.01 g; 1% ZnSO4 , 1 ml; 0.5% CuSO4 , 1 ml; Agar, 20.0 g; AZCL--glucan, 2.0 g (MegaZyme, Australia)). The germinated spore isolates were then exposed to short wave (254 nm) UV germicidal lamp (model 782 L30, Westinghouse Electric Company, Pittsburgh, PA, USA) at a distance of 30 cm for 0 (control plate), 1, 2.5, or 5 min. Plates were placed at 20 ◦ C, in the dark for the first 48 h to prevent photoreactivation and then transferred to light for an additional 5 days. The -glucanase activity was directly monitored on the 0.2% AZCL--glucan MCD agar medium by measuring the clearing zones/blue halos surrounding each colony. Under a dissecting microscope, single hyphal tip isolations were made aseptically from the colonies showing large clearing zones and transferred onto PDA and 0.2% AZCL--glucan MCD agar. Cultures were grown in full light (12.5 einstein m−2 s−1 ) at 20 ◦ C. Phenotypic changes in colony colour, culture shape, culture size and sporulation were recorded at 14 days post inoculation. In addition, mycelia plugs were removed from the edge of duplicate mutant cultures grown on PDA using cork borer #3 (6 mm in diameter) after 4–5 days incubation at 20 ◦ C under light. Three plugs were placed in 20 ml of MCD broth + 1% “inducer” (ground powder of sclerotia of S. sclerotiorum which is rich in glucan [12]) in a 125-ml Erlenmeyer flask. Cultures were incubated on a shaker at 200 rpm, at 20 ◦ C under continuous light (12.5 einstein m−2 s−1 ) and assayed for extracellular enzyme activity at 3 and 5 days by the modified reducing sugar assay (MRSA, described under Section 2.4). In addition, cultures were also tested on potato dextrose broth (PDB) + 1% “inducer” in the same manner described above. High enzyme-producing cultures were repeatedly sub-cultured on MCD broth supplemented with 0.2% AZCL--glucan,
2.3. Method B: UV irradiation of pycnidiospores in suspension Pycnidiospores of C. minitans strain 2134 were harvested from the surface of 14-day-old PDA cultures and suspended in 0.2% barley -glucan MCD broth (l−1 : NH4 H2 PO4 , 2.0 g; K2 HPO4 , 1.0 g; MgSO4 ·7H2 O, 0.5 g; KCl, 0.5 g; FeSO4 , 0.01 g; 1% ZnSO4 , 1 ml; 0.5% CuSO4 , 1 ml; and barley -glucan, 2.0 g (MegaZyme, Australia)) to a final concentration 1×104 pycnidiospores ml−1 . A 10 ml volume of suspension was placed in an open Petri dish (8.7 mm diameter) and exposed to a short wave UV light germicidal lamp (model 782 L30, Westinghouse Electric Company, Pittsburgh, PA, USA) at a distance of 10 cm. A 1 ml control sample was removed from the plate prior to UV exposure. To identify the optimal length of UV exposure, samples were removed after 1, 3, and 5 min. The length of exposure to UV was then adjusted to 3 min for all subsequent experiments as this time was found to kill 97–99% of the pycnidiospores. After irradiation 200 l of spore suspension was transferred into each well of a microplate (or 1 × 103 spores per well and approximately 10–30 viable spores per well). Plate column 1 was used as a control lane, which contained 200 l of non-irradiated spores. Plates were placed at 20 ◦ C in the dark for the first 48 h to prevent photoreactivation and then transferred to full light at 20 ◦ C for an additional 14 days. To select constitutive mutants for -glucanase activity, pycnidiospores were UV irradiated in an identical method as described above, except the suspensions were prepared in PDB. A 50 ml sample of spent culture supernatent taken from each well of the culture plate, was screened by the microplate diffusion assay (MDA) for high -glucanase activity as previously described [9,13]. The commercially available -1,4-endoglucanase (EC 3.2.1.4, Megazyme) at concentrations of 2, 1, 0.5, 0.25, 0.125, 0.06 and 0.00 U ml−1 (diluted in 25 mM Na acetate, pH 4.5) were used as standards. The amount of mycelia present per well of the culture plate was estimated by reading the optical density at a wavelength of 650 nm [14]. The wells with relatively high enzyme levels were transferred onto 0.2% AZCL--glucan agar plates. After 3–4 days individual hyphal tips (10/plate) were isolated under a dissecting microscope from the colony, transferred onto PDA in Petri dishes, and incubated at 20 ◦ C under light for 4–5 days. Single hyphal tip cultures that differed phenotypically, were selected and tested for enzyme production. Mycelia plugs were removed from the edge of the mutant cultures on PDA using cork borer #3, and placed in 3 ml of MCD broth + 1% “inducer” medium in 15 ml test tubes with three plugs per tube. The test tubes were placed on an angle in a test tube rack on a shaker, and incubated at 20 ◦ C, in full light while shaken at 200 rpm. At 7 and 14 days, cultures were tested for enzyme activity using the MDA [9,13] described above. The highest -glucanase-producing
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C. minitans strains were stored in liquid nitrogen, and single hyphal tips were sub-cultured, transferred to PDA/0.2% AZCL--glucan MCD agar and transferred to PDA to allow phenotypic characterization (described below). To confirm the -glucanase production, cultures were again grown in 3 ml of MCD broth + 1% “inducer” and again retested using the MDA. The highest enzyme producing cultures were then grown in 10 ml of MCD broth + 1% “inducer” per 50-ml flask, for 7–14 days and tested for -1,4:1,3-glucanase activity using the MRSA (described below). Constitutive mutants were screened on PDB. Enzyme production of mutant strains was compared to the enzyme activity of the parental strain 2134. 2.4. Characterization of mutants 2.4.1. Enzyme production Extracellular enzyme production was measured from C. minitans wild type strain 2134 and derived mutants (M11-3B2, A10-4, and A8-1). They were grown in PDB, PDB + 1% “inducer”, and MCD broth + 1% “inducer”. Three mycelia plugs from each strain, were removed using cork borer #3 from the edge of 5-day-old colonies on PDA, and used to inoculate 50 ml of broth in a 250-ml Erlenmeyer flask. Fungal cultures were grown in shake cultures (200 rpm) at 20 ◦ C under continuous light (12.5 einstein m−2 s−1 ). Cultures were sampled at 3, 6, 9, 12, and 15 days after inoculation by aseptically removing 1 ml of growth media from each flask. Samples were collected in a 1.5 ml Eppendorf tubes and centrifuged at 5000 × g for 5 min. Extracellular enzyme activities of C. minitans cultures grown in PDB were measured in sample supernatant by the MDA [9,13] and Ostazin Brilliant Red-hydroxyethyl cellulose (OBR-HEC, Sigma #O-6879) [15]. The MDA was used to screen for high -1,4-glucanase (EC 3.2.1.4) utilized 0.2% AZCL-HE-cellulose (Megazyme). Assays were performed in duplicate. Cultures grown on MCD media were assayed using the MRSA. Assays were performed in a 96-well titration plate [16]. Fifty microliters of sample supernatant/enzyme was added to each well and equilibrated to 37 ◦ C. Then, 50 l of carboxmethylcellulose (#C-5013 Sigma–Aldrich Corp., St. Louis, MO, USA), lichenan (Sigma #L-6133), laminarin (Sigma #L-9634) or -glucan (Megazyme-P-BGBH) diluted in 25 mM sodium acetate, pH 4.5, at a concentration of 5 mg ml−1 and equilibrated to 37 ◦ C was added to the enzyme. Assays were allowed to react for 15 min at 37 ◦ C and were stopped by the addition of 100 l of cold solution B (0.3% 3,6-dinitrophthalic acid) and solution C (1.8 M K2 CO3 , +0.1 M Na2 S2 O3 ) mixed prior to use (1:1 v/v). Samples were heated for 45 min at 95 ◦ C, until colour change was completed. The absorpbance was measured at 410 nm using a plate reader (SLT instruments, 16895, Tecan US Inc., Durham, NC, USA). Background absorpbance was measured by a control sample subtracted from the sample
absorpbance. Control reactions were prepared for each sample, following the same method outlined above for the test assays without incubation. One unit of enzyme activity was defined as the amount of enzyme required to release reducing sugars equivalent to 1 mol of glucose per min per ml at 37 ◦ C. 2.4.2. Dry matter weight The solid mass formed in the fungal cultures (45 ml after sampling) were collected at 15 days after growing on PDB, PDB + 1% “inducer”, and MCD broth + 1% “inducer” and centrifuged at 5000 × g for 10 min. Mycelia pellets were transferred into aluminium foil measuring dishes and heated for 48 h at 90 ◦ C. The remaining dry mass in each plate was weighed. 2.4.3. Phenotypic characterization To characterize the C. minitans parental and mutant cultures, a cork borer #3 was used to remove agar plugs containing mycelial mats from the edge of 4–5 days old, PDA cultures. The mycelia plugs were placed on PDA, 0.2% AZCL--glucan supplemented PDA (for constitutive mutants), 0.2% barley -glucan MCD agar, or 0.2% AZCL--glucan MCD agar plates with one plug at the centre of each Petri dish. Cultures were grown in full light (12.5 einstein m−2 s−1 ) at 20 ◦ C. Phenotypic changes in colony colour, culture shape, culture size and sporulation were recorded at 14 days post inoculation.
3. Results and discussion 3.1. Method A: UV irradiation of pycnidiospores on agar The results obtained from method A demonstrated the feasibility of selection of C. minitans strains for enhanced -glucanase production by UV irradiation. UV irradiation was used in this study because of its simplicity, however, the use of other mutagens such as nitrosoguanidine (NTG) may improve the rate of mutant production. Approximately, 50 colonies of C. minitans mutants were obtained from the wild type strain 2134 through UV irradiation. The survival rate of pycnidiospores of C. minitans was approximately 88, 67 and 50% after UV irradiation for 1, 2.5 and 5 min, respectively. Approximately, 50% of the single spore cultures that survived UV irradiation produced clearing zones of greater than 3 mm after 7 days on 0.2% AZCL--glucan MCD agar. These cultures were further purified by single hyphal tip isolations onto PDA and 0.2% AZCL--glucan MCD agar. The primary mutant culture, M11 produced by 1 min exposure to UV irradiation, was determined to have relatively high -glucanase production when screened on 0.2% AZCL--glucan MCD agar. Culture M11-3 (1.88 U ml−1 ), a single spore culture derived from original M11 mutant was about five times higher for -glucanase expression than the
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wild type strain 2134 (0.33 U ml−1 ) after 5 days in MCD broth + 1% “inducer”. In an attempt to further increase -glucanase production by M11-3, it was continuously sub-cultured on 0.2% AZCL--glucan MCD agar. Mutant M11-3B2 was a descendant of the original mutant M11 following eight passages on 0.2% AZCL--glucan MCD agar. Difference in morphological characteristics between mutant M11-3B2 and wild type strain 2134, were evident in cultures grown on PDA for 14 days at 20 ◦ C under light. Colonies of C. minitans wild type strain 2134 have golden-brown pigmentation, smooth edges, and are spore-producing. Colonies of C. minitans mutant strain M11-3B2 have olivaceous golden-brown pigmentation, smooth edges and are spore forming and grew at a faster rate than 2134. Mutant strains derived from the original mutant M11 were unstable when grown on PDA for extended periods, resulting in a decline in enzyme activity. Therefore, passage on a minimal medium such as 0.2% AZCL--glucan MCD agar was essential for culture maintenance.
and screened for -glucanase expression by the MDA. Constitutively expressing -glucanase cultures were identified by measuring -glucanase activity in PDB. Strains A8-1 and A10-4 were identified as constitutive -glucanase producing mutants. Colonies of strain A8-1 grown on PDA at 20 ◦ C under light have yellowish-white pigmentation, serrated edges, and produce very few spores where as colonies of A10-4 have yellowish white pigmentation, serrated edges and produce very few spores. Wild type strain 2134, when cultured on PDA supplemented with 0.2% AZCL--glucan, did not produce a visible zone of clearing surrounding the colony (Fig. 1). Strain 2134, will not produce -glucanase until after the starches and simple sugars have been utilized. The mutant strains, A10-4 and A8-1 with constitutive expression have a visible zone of clearing surrounding the fungal colony. Measuring from outer mycelia edge of the colony, their clearing zones were approximately 5–7 mm wide. Mutant M11-3B2 had a smaller zone of clearing (approximately 3 mm wide).
3.2. Method B: UV irradiation of pycnidiospores in suspension
3.3. Fungal growth and enyzme production
Method B increased the speed of mutant screening and selection. Approximately, 6000 viable UV irradiated pycnidiospores of C. minitans wild type strain 2134 were screened using 0.2% -glucan MCD broth as the initial growth medium. In this approach, mutated spore cultures having a mutation up-regulating -glucanase activity, should grow faster and out compete mycelia from spores with a mutation down-regulating -glucanase activity or no mutation for -glucanase activity. Using the single hyphal tip isolation method, 75 mutant strains were obtained from the UV-treated cultures, which showed relatively high -glucanase activities in the screening tests of microplate wells. These mutants were twice tested for -glucanase production by growing in 3 ml of MCD broth + 1% “inducer”. Of these mutants, 26 mutants were re-tested a third time for enzyme production by growing cultures in a larger 10 ml volume of MCD broth + 1% “inducer” in 50-ml Erlenmeyer flask. The highest enzyme-producing mutant was the mutant strain A7-3D. Colonies of strain A7-3D grown on PDA at 20 ◦ C under light for 14 days have golden-brown pigmentation, serrated edges and produce spores. Approximately, 4000 UV irradiated viable pycnidiospores of C. minitans wild type strain 2134 were grown in PDB and screened for -glucanase activity. We selected for constitutive mutants by using PDB as the selection medium, as non-constitutive fungal isolates would be repressed by simple sugars present in the PDB. Of the 352 wells screened two wells gave relatively high -glucanase activity with relatively low amounts of mycelia present in the well. The mycelia from these wells were transferred and grown on PDA. Single hyphal tip isolations were made from the colony to establish pure cultures on PDA. The cultures derived from these two wells were grown on 3 ml PDB test tube cultures
The average dry mycelial weight showed that each of the four strains of C. minitans grew the best in PDB + 1% “inducer”, followed by PDB and then MCD broth + 1% “inducer” (Table 1). PDB is rich in nutrients (starches and simple sugars). The 1% “inducer” is a natural source of carbohydrate, primarily consisting of -glucan [12]. As expected, the cultures grew best in PDB + 1% “inducer” due to the greater amount simple carbohydrate present. The wild type strain 2134 grew faster in PDB than the mutant strains, which therefore correlated with the larger colony size of strain 2134 on PDA. The characterization of enzyme expression by the wild type strain 2134 and its mutants M11-3B2, A10-4, and A8-1 in various media demonstrated the importance of induction and repression in the control of -glucanase expression. The barley -glucan and cellulose hydrolyzing activity in wild type strain 2134 was greatly repressed by glucose and simple sugars when grown in PDB, producing insignificant amounts of extracellular -glucanase and cellulase. While, mutants A10-4 and A8-1, both selected on PDB, were not repressed by the presence of glucose in PDB and mutant M11-3B2 was only weakly repressed. Mutant A8-1 produced an average 2.74 ± 0.55 U ml−1 of barley -glucan hydrolyzing activity in PDB after 15 days and mutant A10-4 produced an average 2.21 ± 0.34 U ml−1 . Similarly, mutants A10-4 and A8-1 produce extracellular cellulose hydrolyzing activity, assayed at 0.90 ± 0.07 U ml−1 by 6 days and 0.91 ± 0.12 U ml−1 by 9 days, respectively. Mutant M11-3B2 produced lower levels at 0.22 ± 0.03 U ml−1 cellulose hydrolysing activity by 9 days. Wild type strain 2134 produced its highest amounts of enzyme activity when grown on MCD broth supplemented only with “inducer” (Table 2). The “inducer” (ground sclerotia of S. sclerotiorum) enhanced -glucanase expression
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Fig. 1. C. minitans wild type strain 2134 and its mutant strains grown under continuous light at 20 ◦ C for 14 days on PDA supplemented with 0.2% AZCL--glucan. Clearing zones around fungal colonies demonstrate constitutive -glucanase activity. Clearing zones were measured from the outer mycelia colony edge to the outer edge of the clearing. Top row from left to right: 2134, A10-4 and A8-1. Bottom row from left to right: A7-3D, M11-3 and M11-3B2.
in the wild type strain 2134 and mutant M11-3B2 when supplemented into media such as MCD or PDB that is low in -glucan content. When strain 2134 was grown in PDB with 1% “inducer” it began to produce cellulase after a 6–9-day delay, exhibiting approximately 0.22 ± 0.01 U ml−1 by 12 days. When, strain 2134 was grown in MCD broth + 1% “inducer” by 15 days it produced 3.35 ± 0.40 U ml−1 of cellulose hydrolyzing activity while the mutant M11-3B2 only had 2.38 ± 0.16 U ml−1 of cellulose hydrolyzing activity. These results substantiate reports that the expression of genes encoding cell wall degrading enzymes from mycoparasitic fungi, is enhanced by the addition of autoclaved mycelium, fungal cell wall extracts, or polymers, such as laminarin and chitin [11,17,18]. It is generally thought that small diffusible molecules derived from host cell walls trigger greater enzyme expression. Interestingly, the “inducer” did not enhance cellulase expression in constitutive mutants, A10-4 and A8-1, suggesting genes and/or regulatory elements associated with cellulase induction were also mu-
tated. Physiological stress and carbon starvation may also be involved in the up regulation of some of the genes encoding fungal cell wall degrading enzymes [18–20]. Mutant M11-3B2, which was weakly repressed, utilized the simple sugars present in the PDB media quicker than the wild type strain 2134 and cellulase expression was greatly enhanced by the presence of “inducer”. Wild type stain 2134 required “inducer” to weakly overcome the glucose and simple sugar repression. The results of our study, suggest a multiple regulatory mechanisms involved in cellulase and -glucanase expression and that repression and induction mechanisms act independently of each other. Only the cultures grown in MCD broth + 1% “inducer” could be analysed by the MRSA, as cultures grown in PDB based media have high reducing sugar levels that result in high background values. The results of the MRSA are shown in Table 2, using laminarin (a) barley -glucan (b) lichenan (c) and CMC (d) as substrates. The CMC substrate is made of only -1,4-d-glucan. The laminarin substrate is
Table 1 Comparison of dry mycelia weighta of C. minitans: wild strain 2134 and its mutant strains A8-1, A10-4, and M11-3B2, grown in liquid mediab Growth medium
PDB PDB + I MCD + I a b
Dry mycelia weight (g per culture) 2134
A8-1
A10-4
M11-3B2
0.61 ± 0.16 0.65 ± 0.12 0.42 ± 0.13
0.43 ± 0.04 0.70 ± 0.06 0.46 ± 0.07
0.45 ± 0.03 0.80 ± 0.03 0.48 ± 0.03
0.54 ± 0.06 0.87 ± 0.25 0.50 ± 0.014
Average dry mycelial weight from 45 ml cultures. PDB (potato dextrose broth), PDB + I (potato dextrose broth + 1% “inducer”) or MCD + I (MCD broth + 1% “inducer”).
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Table 2 Comparison of production of extracellular -glucanases by C. minitans parental strain 2134 and its mutant strains, A8-1, A10-4 and M11-3B2 after 15 days post inoculation Strains
Enzyme activity (U ml−1 ) Cellulasea PDB + Ie
A8-1 A10-4 M11-3B2 2134
0.29 0.16 2.04 0.21
± ± ± ±
0.01 0.05 0.05 0.01
MCD + Ie 0.04 0.91 2.38 3.35
± ± ± ±
0.02 0.11 0.16 0.40
Laminaraseb
-Glucanasec
Lichenased
MCD + Ie
MCD + Ie
MCD + Ie
5.33 4.33 7.22 5.99
± ± ± ±
0.14 1.1 0.49 0.29
5.48 6.51 6.50 6.00
± ± ± ±
0.29 0.35 0.35 0.04
4.86 4.26 6.51 4.46
± ± ± ±
0.42 0.37 0.15 0.21
a Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: cellulose for cellulase activity (-1,4-endoglucanase activity). b Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: laminarin for laminarinase activity (-1,3-endoglucanase activity). c Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: barley -glucan for barley -glucanase activity (-1,3 and 1,4-endoglucanase activity). d Enzyme activity of the supernatant from cultures were measured by the MRSA for activity on the following substrates: lichenan for lichenase activity (-1,3 and 1,4-endoglucanase activity). e Culture media: C. minitans strains were grown in either PDB + 1% “inducer” (PDB + I), or MCD broth + 1% “inducer” (MCD + I). Cellulose hydrolyzing activity is measured by the OBR-HEC assay method for PDB + I, and by the MRSA for MCD + I.
made of only -1,3-d-glucan. Lichenan and barley -glucan consists of both 1,3 and 1,4 -d-glucan. Wild type strain 2134 produced the highest level of cellulose hydrolyzing activity (3.35 ± 0.40 U ml−1 ) while the mutant M11-3B2 produced the highest level of laminarin hydrolyzing activity (7.22 ± 0.49 U ml−1 ) by 15 days. Also, M11-3B2 mutant strain produced more lichenan hydrolyzing activity (6.50 ± 0.35 U ml−1 ) and similar amounts of barley -glucan hydrolyzing activity (6.51 ± 0.15 U ml−1 ) as the wild type strain 2134 (4.46 ± 0.21 and 6.00 ± 0.04 U ml−1 , respectively). The substrate lichenan has a higher ratio of -1,3 to -1,4 linkages than barley -glucan, thereby explaining the slightly enhanced barley -glucan hydrolyzing activity of strain 2134. Mutants A8-1 and A10-4 produced very low levels of cellulase, but still produced 4.33±1.1 and 5.33 ± 0.14 U ml−1 , respectively of laminarin hydrolyzing activity which was only slightly lower than strain 2134. Various applications have been suggested for the utilisation of cell wall degrading -glucanase activity, such as their use in agriculture as feed additives [21] or their production by superior strains of C. minitans as biocontrol agents [22]. A mutated C. minitans strain with superior -glucanase activity could ultimately be used to produce -glucanase on an inexpensive crude substrate such as a food processing waste product. For large scale economic fermentation of C. minitans strains having -glucanase activity, it is desirable to have constitutive -glucanase activity that does not require induction by a source of -glucan and which is not repressed by the presence of simple sugars. Although natural sources of -glucan are available, such as the “inducer” obtained from the sclerotia of S. sclerotiorum, many inexpensive and readily available growth media, such as potato skins, do not provide a -glucan source. It is preferable not to have to add a -glucan source to the culture media. The creation of stable C. minitans mutants with enhanced
and/or constitutive -glucanase expression is the first step in: developing effective mutagenesis, screening and selection methods; increasing our knowledge and understanding of constitutive expression; maximising -glucanase expression; and developing good candidate fungal strains for use in potential commercial enzyme fermentation systems.
4. Conclusions From this study, it is concluded that UV irradiation can induce C. minitans mutants that are culturally stable. The control of -glucanase expression is multigenic and intricate. Selective screening methods such as initial growth on PDB or 0.2% barley -glucan MCD broth followed by the MDA can be applied successfully to isolate specific mutants with constitutive and/or enhanced -glucanase expression.
Acknowledgments The authors would like to thank J. Yanke for maintaining the stock cultures of C. minitans for this study at the Culture Collection Centre, Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alta., Canada. References [1] Stone BA, Clarke AE. Chemistry and biology of (1–3)--glucans. Melbourne, Australia: La trobe University Press; 1992. [2] Wessels JGH, Seitsma JH. Fungal cell walls: a survey. In: Tanner W, Loewus FA, editors. Encyclopedia of plant physiology 13B. Berlin: Springer; 1981. p. 352–94. [3] Huang HC, Hoes JA. Penetration and infection of Sclerotinia sclerotiorum by Coniothyrium minitans. Can J Bot 1976;54:406–10.
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