Applications of Enzymes to Lignocellulosics - American Chemical

Chapter 18

Downloaded by COLUMBIA UNIV on July 19, 2012 | http://pubs.acs.org Publication Date: August 7, 2003 | doi: 10.1021/bk-2003-0855.ch018

Amplification of Cellulase Genes and Cellulase Hyperproducers in Trichoderma: Minireview H . Toyama, T . Hotta, N . Yamagishi, and N . Toyama Department of Food Science and Technology, Faculty of Horticulture, Minamikyushu University, Takanabe, Miyazaki 884-0003, Japan

Nuclear diameter in conidia and mycelia of Trichoderma reesei could be enlarged by a mitotic arrester, colchicine. This result means that chromosomes, including cellulase genes can be amplified by such reagent. Using this reaction, we constructed cellulase hyperproducers of this fungus. A haploidizing reagent, Benomyl, was used in order to carry out chromosomal (genetical) recombination. As the primary selection, double layer selection medium including selection substrates, Avicel, wood powder, or absorbent cotton contributed to selecting hyperproducers. As the secondary selection, Avicel liquid medium test could be used. In this report, we demonstrate the nuclear changes by colchicine treatment and the consequent pathway of selection of cellulase hyperproducers in Trichoderma.

Trichoderma is known producer of stable cellulase in high yields, and is widely utilized for industrial cellulase production (1,2). The cellulasefromthis fungus is also widely used for processing of foods such as baby foods ( 3 ). This enzyme also contributes to recycling of cellulosic wastes ( 4 ). However, the cellulase productivity of this fUngus is inadequate for many purposes and productivity needs to be increased ( 5 ). Colchicine is a well-known mitotic arrester and is widely used for plant breeding ( 6 ). In the breeding of

304

© 2003 American Chemical Society In Applications of Enzymes to Lignocellulosics; Mansfield, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

305 Trichoderma, chemical mutation techniques and / or genetic engineering techniques are usually applied (7,8). Therefore, we attempted to construct an alternative breeding system for this fungus using autopolyploidization and haploidizing techniques.

Autopolyploidization


Colchicine treatment of normal conidia Dried green mature conidia (mononucleate) of Trichoderma were incubated statically in Mandels' medium containing 1.0% (w/v) glucose (Wako), 0.5% (w/v) peptone (Difco), and 0.1% (w/v) colchicine (Wako) (pH 6.0) at 28°C. Nuclear changes were observed by nuclear staining with Giemsa and DAPI solutions. During colchicine treatment, the diameter of the nuclei continued to increase (9 ). After about 7 days of incubation, multiple smaller nuclei were generated from the single larger nucleus (autopolyploid nucleus) in a conidium. It is suspected that one larger nucleus was collapsed in a narrow conidium followed by the generation of multiple smaller nuclei. As a result, a multinucleate conidium was produced. This phenomenon is called multinucleation. When the colchicine treatment was prolonged, multiple minute nuclei were generatedfromnuclei in a conidium. We call this micronucleation.

Colchicine treatment of swollen conidia Dried green mature conidia were incubated in Mandels* medium containing 1.0% glucose and 0.5% peptone (pH 6.0) for 10 h at 28^ using a rotary shaker to prepare swollen conidia (10). These swollen conidia were incubated without shaking in Mandels medium containing 1.0% glucose, 0.5% peptone, and 0.1% colchicine (pH 6.0) at 28°C. As the inner volume of these swollen conidia was larger than that of the original conidia, larger autopolyploid nuclei could be produced in the swollen conidia. The nuclear changes in a swollen conidium were the same as those in the original conidium during colchicine treatment. Multinucleation and micronucleation also occurred. 1

Colchicine treatment of the mycelial mat A mycelial mat (10 mm x 10 mm) of this fungus was incubated without shaking in Mandels medium containing 1.0% glucose, 0.5% peptone, and 0.1% colchicine at 28°C, and nuclear changes were observed by nuclear staining with Giemsa solution and DAPI solution. Autopolyploidization, multinucleation, and micronucleation were also observed in the mycelia during colchicine treatment. Many nuclei appeared when multinucleation occurred in the mycelia ( 11 ). 1

In Applications of Enzymes to Lignocellulosics; Mansfield, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

306 Therefore, we called this structure a polykaryon. The nuclear diameter of autopolyploid nuclei was not uniform because these nuclei were not synchronized before the experiments.


Multinucleation Multinucleation occurred when conidia or mycelia containing autopolyploid nuclei were treated with chemical reagents, e.g., higher concentrations of colchicine (2%), coumarin (Wako), or trypan blue (Wako), and when such conidia or mycelia were incubated at higher temperatures (12). Micronucleation also occurred on prolongation of this treatment. However, multinucleation was distinguishedfromhaploidization because it was unknown whether genetic recombination occurred.

Haploidization Benomyl treatment of swollen conidia in the liquid medium When the swollen conidia containing autopolyploid nuclei were incubated without shaking in Mandels' medium containing 1.0% glucose, 0.5% peptone, and 0.6 /x g/ml benomyl (Sigma) (pH 6.0), multiple smaller nuclei were generated from the single larger nucleus (autopolyploid nucleus) in a swollen conidium. The diameter of such smaller nuclei decreased when the treatment time was prolonged. White colonies appeared with a high frequency on the medium when such conidia were spread on a potato dextrose agar (PDA) medium containing 0.1% (v/v) Triton X-100 (Wako) followed by incubation at 28°C. Benomyl treatment of swollen conidia on the solid medium There were few white colonies when the swollen conidia containing autopolyploid nuclei were incubated on a PDA medium containing 0.1% Triton X-100 and 0.6 \x g / ml benomyl (pH 6.0) at 28°C. Almost all of the colonies generated green conidia.

Benomyl treatment of mycelial mat on the solid medium Fan-shaped sectors were produced around the colony when a mycelial mat containing autopolyploid nuclei was incubated on the PDA medium containing 0.6 ii g / ml benomyl (pH 6.0) at 28°C (13 ). Some sectors generated white


307

conidia. The diameter of nuclei in these sectors was almost the same as that of the original strain.

Successive autopolyploidization and haploidization


When a haploidized mycelial mat ( a fan-shaped sector) was incubated in Mandels' medium containing 1.0% glucose, 0.5% peptone, and 0.1% colchicine again at 28°C, autopolyploidization occurred again, and the nuclear diameter also increased. Haploidization could be also carried out on such mycelial mats

Nuclear changes and cellulase productivity Changes in cellulase productivity of colchicine-treated conidia

Green mature conidia were incubated in 0.001% or 0.01% (w/v) colchicine solution for 24 h at 28°C using a reciprocal shaker (125 strokes / m). These treated conidia were collected and spread on PDA medium containing 0.1% Triton X-100 followed by incubation for 4 days at 28°C. Two colonies that produced a deeper yellow pigment were isolated. One yielded mononucleate conidia and the other binucleate conidia. The colony with mononucleate conidia was named strain A and the binucleate conidia colony was named strain B. The conidia derived from A and B were re-treated with 2.0% colchicine for up to 10 days. Colonies were isolated by plating treated conidia on PDA plates containing 0.1% Triton X-100 followed by incubation for 4 days at 28°C. Two colonies that generated multinucleate conidia were selected from among 26 (strain A) and 32 (strain B) colonies, respectively, by nuclear staining. The colony derived from strain A was named strain C and the other derived from strain B was called strain D. One loopful of conidia was grown in 50 ml of Mandels' medium containing 1.0% (w/v) Avicel and 0.5% (w/v) peptone (pH 5.0) in a 100-ml Erlenmeyer flask for 5 days using a rotary shaker (160 rpm) at 30°C. Mycelia were then removed on a 3G-3 glass filter and the filtrate was used as the source of cellulase. Avicel-hydrolyzing activity, CMC-hydrolyzing activity, and Salicin-hydrolyzing activity were measured using 1.0% Avicel, 1.0% CMC-Na (carboxymethylcellulose sodium-salt) (D.S. 0.7), and 1.0% Salicin (Wako), respectively, suspended in 0.1 M acetate buffer (pH 5.0) as substrates. Enzymes (2ml) and substrates (4ml) were mixed and incubated for 60 min at 40°C. The enzyme reaction was then stopped by addition of 0.1 N HC1. The amount of glucose generated was measured using the Glucose-test Wako (Wako). Enzyme activity was defined as the amount of enzyme that produces 1 /x mol of glucose per min. Avicel hydrolyzing activity, CMC hydrolyzing activity, and Salicin hydrolyzing activity were compared among the conidial variants (strain A, B, C,


308 and D). The cellulase productivity of the conidial strains A increased more than that of B and the productivity increased in both C and D as shown in Table 1 (15).


Changes in cellulase productivity of colchicine-treated mycelia We attempted to select the strains with higher Avicel degrading activity from among the conidia of the haploidized colony derived from colchicinetreated mycelial mat using the double-layer selection medium (for primary selection) and Avicel liquid medium (for secondary selection) (16). For the primary selection, 96 ml of the Mandels medium containing 1.0% Avicel, 0.5% peptone, 1.0% (w/v) substrate, 0.1% Triton X-100,1.5% agar, and conidia (the bottom layer medium) was added to a deep glass plate (150 mm in diameter and 60 mm in depth) and left at 4°C in order to harden the agar. After hardening agar, 196 ml of Mandels' medium containing 1.0% Avicel, 0.5% peptone, 1.0% substrate, 0.1% Triton X-100, and 1.5% agar (the upper layer medium) was added on the bottom layer medium followed by hardening of the agar at 4°C. Avicel, wood powder (Fagus crenata), and absorbent cotton (for medical use) were used as the substrates for selection. Ten loopfuls of the conidia generated on the haploidized colony were incubated in the medium for primary selection for 6 days at 28°C. The colonies which could break thorough the selection layer were used for the secondary selection. Ten colonies were selected on the selection medium containing Avicel, four colonies were selected on the selection medium containing absorbent cotton, and four colonies were selected on the selection medium containing wood powder. The Mandels' medium containing 1.0% Avicel and 0.5% peptone was used (pH 5.0) as the Avicel liquid medium for the secondary selection. A 1

mycelial mat (2 mm x 2 mm) of the colonies selected by the primary selection was added to the medium for secondary selection and incubated by a rotary shaker (TAITEC NR-30, Koshigaya, Japan) (80 rpm) at room temperature (16 23°C). After the Avicel liquid medium became transparent, the amount of Avicel sedimentation was observed by leaving it for 1 h. T. reesei Rut C-30 took 6 days to make the Avicel liquid medium transparent whereas strains selected by Avicel, AV-1 and AV-2, took 4 days to make the medium perfectly transparent as shown in Fig. 1. The strain selected by absorbent cotton, AB-1, and those selected by wood powder, BU-1 and BU-2 could also make the medium transparent after 4 days of incubation. All of the selected strains were compared with T. reesei Rut C-30 in cellulose hydrolyzing activity by wheat bran culture. A mycelial mat (2 mm x 2 mm) of the selected strains was added to flasks of the wheat bran medium and incubated at 28°C for 6 days and these flasks were shaken once a day. After incubation, 15 ml of 0.1 M acetate buffer (pH 5.0) was added, stirred using a glass rod, and left to stand for 1 h. The enzyme solution was then extracted from the wheat bran culture using a nylon cloth. The extracts were



4.4

14.6

18.9

Conidia B

Conidia C

Conidia D

12.2

11.6

SOURCE: Reproduced from Reference 15. Copyright 1995 Society for Industrial Microbiology

with 2.0% colchicine for 10 days at 28°C (multinucleate).

treatment with 2.0% colchicine for 10 days at 28°C (multinucleate). D was derived from the conidia B after treatment

the conidia treated with 0.01% colchicine for 24 h at 28*0 (binucleate). C was derived from the conidia A after

was derived from the conidia treated with 0.001% colchicine for 24 h at 28°C (mononucleate). B was derived from

NOTE: One loopful of conidia was added to cellulose medium and the activity was measured after 5 days at 30°C. A

18.8

12.3

9.6

10.0

10.4

7.4

Conidia A 2.2

2.2

4.4

(IU / ml)

hydrolyzing activity

Salicin

5.9

(IU / ml)



(IU / ml)

CMC

Avicel

Original strain

Conidia

Table 1. Cellulose hydrolyzing activity in conidial strains


310

Figure 1. Results of secondary selection using Avicel liquid media test OLapsetime 0 Rut C-30

1

2

3

4

=====

5

6

—=>


Strains selected by Avicel AV-1 AV-2 AV-3 AV-4 AV-5 AV-6 AV-7 AV-8 AV-9 AV-1Q

=

=

— =

— = =

=--

-=> —> -==> => => > _ =

—

> > —=> -=>

Strains selected by Absorbent cotton AB-1 = = = = = _ = > AB-2 AB-3 AB-4

===============================> = = = = = = = = — = = — — — >

Strains selected by Wood powder BU-1 ======™===========> BU-2 = = -=> BU-3 — > BU-4 — = —=> A mycelia mat (2mm * 2 mm) was added to the liquid Mandels' medium containing 1.0% (w/v) Avicel and 0.5% (w/v) peptone (pH 5.0) and incubated for 6 days at room temperature (16-23°C) suing a rotary shaker (80 rpm). The degree of transparency of the medium and the amount of Avcel sedimentation were compared everyday. The arrows show the time necessary for making the medium transparent (Reproduced with permission from reference 16. Copyright 2002 Humana Press, Inc.)



311

centrifuged at x 5510 g, and the top clear portion was used as the enzyme solution. As substrates for the enzyme reactions, 1.0 g of Avicel, CM-cellulose (Wako), or Salicin (Wako) were added to 99 ml of 0.1 M acetate buffer (pH 5.0) followed by 0.2 ml of enzyme solution and 4.0 ml of substrate mixed, and incubated for 60 min at 40°C using a reciprocal shaker (THOMASTAT T-22S, Tokyo, Japan). The agitation speed was 125 strokes / min. The reaction mixture was filtered with filter paper (no. 2, Whatman). The amount of reducing sugar in the reaction mixture was measured using the Glucose CII test Wako (Wako). IU was based on the amount of enzyme-producing reducing sugar equivalent to 1 M mol of glucose per minute. Although only Avicel-hydrolyzing activity increased in AV-1 selected by Avicel, all activity, Avicel, CMC, and Salicin hydrolyzing activity, increased in AV-2. In the strain AB-1 selected by absorbent cotton, Avicel hydrolyzing activity increased especially as shown in Table 2. In the strains BU-1 and BU-2 selected by wood powder, all Avicel, CMC, and Salicin hydrolyzing activity increased.

Conclusion and consideration for future This report demonstrates that colchicine is effective even in a cellulolytic fungus, Trichoderma. As colchicine has been widely utilized for breeding plants for a long time, this reagent had assumed to be effective only in plants. But, we show here that colchicine is also effective on microorganisms. We have already reported that colchicine can modify nuclei in fungi; Aspergillus kawachii ( 17 ), Basydiomycetes\ Pleurotus ostreatus (18 ), Flammulina velutipes ( 19 ), Lentinus edodes ( 20), and yeast; Saccharomyces cerevisiae (21 ). As

colchicine is also active on Basydiomycetes, which can digest lignin, further investigation should be also carried out on such organisms. Moreover, one of the merits of this autopolyploidization technique is that many and various genes can be amplified simultaneously. So, this advantage should be also utilized effectively in the production of Trichoderma strains for specific applications. From the results described here, we conclude that these autopolyploidization and haploidization techniques are effective on breeding of the fungus, Trichoderma.

References 1. Hayward, T.K.; Hamilton, J.; Tholudur, A.; Mcmillan, J.D. Appl. Biochem.Biotechnol.2000, 84/86, 859.

2. Heikinheimo, L.; Buchert, J.; Miettinen-Oinonen, A.; Suominen, P. Text Res. J. 2000, 70, 969. In Applications of Enzymes to Lignocellulosics; Mansfield, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


35

Rut C-30

45 49 16

41 55 12 24 22 45 32

37

32

30

30

28

24

16

AV-4

AV-5

AV-6

AV-7

AV-8

AV-9

AV-10

26 22

39

36

AB-2

AB-3

28

53

AB-1

Strains selected by absorbent cotton

22

39

47

AV-3

22

26

30

26

20

39

35

57

47

49

AV-2

20

Salicin

22

28

CMC

30

75

AV-1

Strains selected by Avicel

Avicel

Strains

and wood powder (IV/ml)

Table 2. Cellulose hydrolyzing activity of the strains selected by Avicel, absorbent cotton,



34

30 23

61 22 24

67

40

28

BU-2

BU-3

BU-4

S O U R C E : Reproduced from Reference 16. Copyright 2002 Humana Press Inc.

wood powder.

N O T E : AV lines were selected by Avicel, A B lines were selected by absorbent cotton, and B U lines were selected by

67

57

34

20

75

16

BU-1

Strains selected by wood powder

AB-4


314

3. Ado, Y. Biosci. Bioind. 1989, 47, 840. 4. Ju, L.-K.; Afolabi, O.A. Biotechnol. Prog. 1999, 15, 91. 5. Sun, T.; Li, Z.; Liu, D. Biotechnol. Tech. 1996, 10, 889.

6. Takayanagi, R.; Kitaura, T.; Mako, M.; Miura, Y. Breed. Sci. 1996, 46, 313. 7. Morikawa, Y.; Kawamori, M.; Shinsha, Y.; Oda, F.; Takasawa, S.; Ado, Y.


Agric. Biol. Chem. 1985, 49, 1869.

8. Durand, H.; Clanet, M.; Tiraby, G. Bioenergy 1985, 84, 246. 9. Toyama, H.; Toyama, N. J. Ferment. Bioeng. 1990, 69, 51. 10. Rosen, D.; Edelman, M.; Galun, E.; Danon, D. J. Gen. Microbiol. 1974, 83, 31. 11. Toyama, H.; Toyama, N. W. J. Microbiol. Biotechnol. 1995, 11, 326. 12. Toyama, H.; Toyama, N. The bulletin of the faculty of horticulture, Minamikyushu univ. 1995; No.25(A), pp 31-36. 13. Toyama, H.; Toyama, N. Appl. Biochem. Biotech. 2000, 84/86, 419.

14. Toyama, H.; Toyama, N. Appl. Biochem. Biotech. 2001, 91/93, 787. 15. Toyama, H.; Toyama, N. J. Indust. Microbiol. 1995, 15, 121. 16. Toyama, H.; Yamagishi, N.; Toyama, N. Appl. Biochem. Biotech. 2002,

17. 18. 19. 20. 21.

98/100, 257. Toyama, H.; Toyama, N. Microbios 1997, 90, 23. Toyama, H.; Toyama, N. J. Biotechnol. 1995, 35, 97. Toyama, H.; Toyama, N. Microbios 1995, 81, 155. Toyama, H.; Toyama, N. Microbios 2000, 101, 73. Toyama, H.; Toyama, N. Acta Biotechnologica 1998, 18, 305.


Applications of Enzymes to Lignocellulosics - American Chemical

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