Báo cáo khoa học: Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials pptx

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials Markku Saloheimo1, Marja Paloheimo1, Satu Ha

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials Markku Saloheimo1, Marja Paloheimo1, Satu Hakola1, Jaakko Pere1, Barbara Swanson2, Eini Nyysso¨nen2, Amit Bhatia2,*, Michael Ward2and Merja Penttila¨1

1

VTT Biotechnology, Finland;2Genencor International, Inc., Palo Alto, CA, USA

Plant cell wall proteins called expansins are thought to

disrupt hydrogen bonding between cell wall

polysaccha-rides without hydrolyzing them We describe here a novel

gene with sequence similarity to plant expansins, isolated

from the cellulolytic fungus Trichoderma reesei The

pro-tein named swollenin has an N-terminal fungal type

cel-lulose binding domain connected by a linker region to the

expansin-like domain The protein also contains regions

similar to mammalian fibronectin type III repeats, found

for the first time in a fungal protein The swollenin gene is

regulated in a largely similar manner as the T reesei

cel-lulase genes The biological role of SWOI was studied by

disrupting the swo1 gene from T reesei The disruption

had no apparent effect on the growth rate on glucose or

on different cellulosic carbon sources Non-stringent

Southern hybridization of Trichoderma genomic DNA

with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant The swollenin gene was expressed in yeast and Aspergillus nigervar awamori Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation

of reducing sugars It also weakened filter paper as assayed by an extensometer The SWOI protein was purified from A niger var awamori culture supernatant and used in an activity assay with Valonia cell walls It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars

Keywords: cellulase; expansin; cellulose binding domain; Trichoderma; regulation

In the last few years, a new class of proteins called expansins

has been discovered in plants (reviewed in [1–3]) A number

of expansin genes have been identified from a wide variety of

plant species, including cucumber, Arabidopsis, rice [4] and

tomato [5] The expansins were first implicated in loosening

the cell wall structure during plant cell growth (the

acid-growth response), and the proteins forming a distinct family

with high sequence identity and having this type of activity

are now classified as a-expansins [6] The group 1 pollen

allergens have approximately 25% amino-acid identity with

a-expansins and have been shown to be active in an

acid-growth assay on grass cell walls Along with their vegetative

homologues they are designated as b-expansins [6]

Expansins have been proposed to disrupt hydrogen bonding between cellulose microfibrils or between cellulose and other cell wall polysaccharides without having hydro-lytic activity [7,8] In this way they are thought to allow the sliding of cellulose fibers and enlargement of the cell wall Purified cucumber expansins have been shown to catalyze extension of isolated plant cell walls such as cucumber hypocotyl walls when assayed using a constant load extensometer [9] These cucumber expansins have also been shown to weaken filter paper without producing reducing sugar [7] Some of the a-expansins are functional during fruit ripening, possibly aiding the action of hydrolytic enzymes that degrade the cell wall polymers [5] Experiments have been made where expansin activity has been over expressed,

or reduced by antisense strategy in Arabidopsis thaliana T he results suggest a role for these proteins in the control of plant growth and morphogenesis [10]

A number of saprophytic and pathogenic fungi and bacteria produce a wide range of enzymes designed to break down plant biomass These enzymes include cellulases that break down cellulose to glucose, and hemicellulases that degrade the different hemicelluloses to monomeric sugars For the degradation of the insoluble and complex plant cell wall the microbes produce multiple enzyme forms belonging

to various enzyme categories For example, from the fungus Trichoderma reesei, one of the best known saprophytic microbes, genes have been cloned that encode two exo-acting cellulases liberating mainly cellobiose from cellulose chain ends, five endo-acting cellulases hydrolyzing internal linkages of cellulose chains and 10 hemicellulases represent-ing different enzyme activities [11,12] Most of the cellulases and some of the hemicellulases of this fungus have a

Correspondence to M Saloheimo, VTT Biotechnology,

PO Box 1500, 02044 VTT, Finland.

Fax: + 358 9 455 2103, Tel.: + 358 9 456 5820,

E-mail: Markku.Saloheimo@vtt.fi

Abbreviations: CBD, cellulose binding domain; CBHI, CBHII,

T reesei cellobiohydrolases; CREI, regulatory protein involved

in catabolite repression in T reesei; EGI, EGII, EGIV, EGV,

T reesei endoglucanases; FnIII repeats, fibronectin III type repeats;

SWOI, T reesei swollenin I; swo1, gene encoding SWOI; HEC,

hydroxyethylcellulose.

Enzymes: endo-1,4-glucanase (EC 3.2.1.4), cellobiohydrolase

(EC 3.2.1.91).

*Present address: EPIcyte Pharmaceutical, Inc., 5810 Nancy Ridge

Drive, Suite 150, San, Diego, CA 92121, USA.

Note: the complete swo1 sequence data has been submitted to the

EMBL database under the accession no AJ245918.

(Received 17 May 2002, revised 3 July 2002, accepted 5 July 2002)

modular structure consisting of a cellulose binding domain

(CBD) at either end of the polypeptide chain, connected to

the catalytic domain with a linker region The role of the

CBD is to mediate binding of the enzyme to the insoluble

substrate

In addition to plants, a protein with an endoglucanase

domain and a domain with sequence similarity to expansins

has been reported in the plant pathogen Clavibacter

michiganensisssp sepedonicus [13] In this paper, we report

the discovery of a novel fungal protein having significant

sequence identity to plant expansins Unlike plant

expan-sins, this protein has a modular structure with an

N-terminal CBD The protein was named swollenin due

to its ability to swell cotton fibers without producing

detectable amounts of reducing sugars

E X P E R I M E N T A L P R O C E D U R E S

Strains, vectors and growth conditions

The T reesei cDNA library in the vector pAJ401 [14] was

screened in the yeast strain H1152 (a, sso2-1, leu2-3, trp1-1,

ura3-1, sso1::HIS3, M Aalto, unpublished results) on

SC-Ura plates with 2% galactose as the carbon source [15] at

the restrictive temperature 31C The yeast strain DBY746

(a, his3D, leu2-3, ura3-52, trp1-289, Cyhr) was used for

swollenin production The T reesei strain QM9414 [16] was

used in Northern studies and for detection of the swollenin

protein For the Northern studies the strain was cultivated

in shake flasks (28C, 200 r.p.m.) in minimal media [17]

containing 5% glucose, 2% sorbitol, 2% cellobiose, 2%

lactose or 2% Solka floc cellulose for 3 days Alternatively,

the strain was grown on 2% glycerol for 72 h, followed by

addition of sophorose (1 mM final concentration) After

further 15 h the culture was harvested A culture grown in a

minimal medium with 2% glycerol for 87 h was used as a

control

Nucleic acid methods

Yeast was transformed with the LiAc method [18] or by

electroporation (Bio-Rad) Plasmid constructs were made

using standard methodology [19] Total T reesei RNA was

isolated as described [20] The RNA samples (5 lg) were

treated with glyoxal and analyzed in a 1% agarose gel

Northern blotting and hybridization were performed on a

Hybond-N nylon membrane (Amersham) T reesei DNA

was isolated as described [21] Stringent Southern

hybrid-ization was performed as described [19] Nonstringent

Southern hybridization was performed in a hybridization

mixture without formamide [19] at 48C and the filter was

washed in 2· NaCl/Cit, 0.1% SDS for 10 min at room

temperature and for 30 min at 48C

Antibodies and Westerns

Swollenin antibodies were generated in rabbits by

immu-nizing with the peptide CDPNYTSSRPQERYGS (amino

acids 422–437 in the swollenin sequence) SDS gel

electro-phoresis was performed as described [22] and Western

blotting was performed on a nitrocellulose membrane

(Schleicher & Schull) and detection of the swollenin with

a secondary antibody-alkaline phosphatase conjugate

(Bio-Rad) Samples of yeast or T reesei supernatants and purified SWOI were denatured for Endoglycosidase H treatment by heating 10 min at 100C in 0.5% SDS, 1% 2-mercaptoethanol and subsequently treated with Endo Hf (New England Biolabs) for 3 h at 37C in the G5 buffer (50 mMNa-citrate, pH 5.5) 1000 units of EndoHfwas used per 20 lg of purified SWOI and per 20 lL of the T reesei

or concentrated yeast supernatants

Swo1 gene disruption The swo1 gene was disrupted from the T reesei strain QM9414 [16] by replacing it with a hygromycin resistance cassette The genomic swo1 gene was first subcloned from a cosmid library clone into pBluescript SK– as a 5.5 kb EcoRV fragment to obtain the plasmid pSH1 Most of the swollenin-coding region was replaced from pSH1 by digesting it with NarI and BstEII and ligating with the hygromycin resistance cassette consisting of the Aspergillus nidulans gpdApromoter and trpC terminator and the E coli hygromycin resistance gene derived from the plasmid pBluekan (from P.J Punt, TNO Nutrition and Food Research, Zeist, the Netherlands) The resulting plasmid, pSH9 was digested with EcoRV and transformed into QM9414 as described [17], and transformants were selected

on 100 lgÆmL)1 hygromycin and purified to uninuclear clones by plating single spores on selective medium Disruptants of the swo1 gene were screened among the transformants by Southern hybridization performed as described [19] Two disruptants obtained were also exam-ined by growing them in shake flasks (28C, 200 r.p.m.,

5 days) in a medium with 3% whey and 1.5% complex grain-based nitrogen source [23] and performing Western analysis from their culture supernatants as described above The phenotype of the disruption was studied by plating single spores on plates with minimal medium [17] supple-mented with 0, 0.1 or 0.2% proteose peptone and either 2% glucose, 2% Solka floc cellulose, 2% Avicel cellulose or 2% complex grain-based carbon/nitrogen source [23] An addi-tional test was made on plates where the minimal medium with or without peptone and without any carbon source had been overlaid by a Whatman 1 filter paper disc Growth of colonies of the swo1 disruptants and the parental strain was followed daily

Production and characterization of the swollenin preparations

The yeast strain DBY746 harbouring a plasmid with swo1

in the vector pAJ401 [14] or the vector alone were grown in Chemap CMF mini 1 L or Biolafitte 14 L bioreactors The bioreactor medium was SC-Ura with 2% glucose as the carbon source [15] The yeast supernatants were concen-trated 20 times with a Centiprep concentrator (Amicon) for the treatments of cotton fibers and filter paper The amount

of SWOI was estimated by comparing signals obtained in Westerns from Endo-H-treated yeast supernatants with signals obtained with known amounts of purified Endo-H treated SWOI from A niger var awamori

The 1.5 kb coding region of the swollenin cDNA clone was amplified by PCR using the following primers which were designed to add BglII and XbaI restriction endonuc-lease sites to the 5¢ and 3¢ ends, respectively Primer

ExAspBgl2: 5¢-CATTAGATCTCAGCAATGGCTGGT

AAGCTTATCCTC-3¢ Primer ExAspXba1:

5¢-CGACTCTAGAAGGATTAGTTCTGGCTAAAC

TGCACACC-3¢ The DNA sequence of the amplified

product was verified and the swollenin coding region was

inserted into the BglI and XbaI sites between the glaA

promoter and terminator of an Aspergillus expression vector

(pGAPT-PG) to produce pGAPT-exp The pGAPT-PG

vector consists of pUC18 containing the A nidulans pyrG

gene as selectable marker and a 1.1 kb fragment of the

A nigervar awamori glaA promoter and a 0.2 kb fragment

of an A niger glaA terminator

The expression plasmid pGAPT-exp was transformed

into A niger var awamori strain dgr246 P2 as described

[24] Transformants were selected for their ability to grow

on minimal medium lacking uridine For swollenin protein

production the transformants were grown in liquid medium

as described [25] Cells were removed and the culture

supernatants were equilibrated with 1M ammonium

sul-phate, 100 mM Tris pH 7 The supernantant was then

applied to a cellulose (Sigma, St Louis, MO, USA) affinity

column and washed with 1Mammonium sulphate, 100 mM

Tris pH 7 to remove unbound proteins The purified

swollenin was eluted as a single peak in water

The purified swollenin was tested for activity against

hydroxyethylcellulose (HEC), b-glucan, xylan and mannan

The enzymatic activities against HEC (Fluka) and barley

b-glucan (Biocon) were determined according to IUPAC

[26] and against birch xylan (Roth) as presented [27]

Mannanase activity was assayed according to the procedure

of IUPAC (1987) but using 0.5% locust bean gum (Sigma)

as a substrate

Action on solid substrates

Yeast supernatants Cotton fibers were mercerized by

treating them with 25% NaOH for 15 min at 5C and

washing several times with distilled water The cotton fibers

were suspended in the concentration of 0.5 gÆmL)1 in

50 mM sodium acetate, pH 5.0 containing 1/4 of the

concentrated yeast culture media from the swollenin

producing yeast and control strain Additionally, the

purified T reesei EG II, CBH I and cellulose binding

domain (CBD) of CBH I at a concentration of 5 lgÆmL)1

were used as controls for the swollenin [28,29] After

incubation for 4 h at 25C, the suspended fibers were

filtered off and the amount of reducing sugars released into

the filtrates was determined as described in [30] The fibers

were rinsed once with buffer and then suspended in distilled

water with glass beads prior to sonication for one minute

using a probe tip sonicator (Vibra Cell Sonics and Materials

Inc.) The fibers were then stained and visualized by light

microscopy to determine gross effects on their structure

For the paper strength test, Whatman no 3 filter paper

was cut into strips measuring 7· 2 cm Sodium acetate

buffer (50 mM, pH 5) was used for all of these experiments

The concentrated yeast samples were sometimes first

desalted by passage through a Bio-Rad Econo-Pac10 DG

column with a molecular mass cut-off of 6000 Da After

desalting, 5 mL of the yeast samples were added to 4 mL of

buffer in 50 mL disposable conical tubes and the Whatman

strips were added At the same time, strips were added to

buffer alone and 8 urea in buffer After incubating at

room temperature for 15 min the strips were measured for their wet tensile strength The assay was performed by placing each wet strip of paper between the Thwing–Albert tensile tester (Model 5564 from Instron Corporation, Canton, MA, USA) clamps spaced 4.5 cm apart A 250 lb load cell was used Test speed was 0.1 cmÆmin)1, and the peak load was measured before breaking; it typically only took a minute to reach the paper breaking load

The purified swollenin The action of the purified swollenin preparation on plant cell wall material was followed using Valonia cell wall fragments as substrate Vesicles of Valonia macrophysa were purified as described previously [31] and cut to small pieces, 4–5 mg each (dry weight) These cell wall fragments were suspended in 50 mMacetate buffer (pH 5.0) and swollenin was added at the dosages of 10 lgÆmg)1and

100 lgÆmg)1 Treatments with the purified T reesei CBH I (also known as Cel7A) and EG II (also known as Cel5A) were performed as comparison for swollenin The samples were incubated at +45C under stirring for 48 h and thereafter examined under a stereo microscope (Leica, Wild M10) The control sample was treated alike but omitting the enzymes and swollenin In addition the filtrates were analysed for solubilized sugars by HPLC

R E S U L T S

Swollenin has sequence similarity with plant expansins The swollenin cDNA was isolated in a screening where compo-nents of the Trichoderma secretory pathway were searched for by yeast complementation The sso2 temperature-sensitive S cerevisiae strain was transformed with a T reesei cDNA expression library, and cDNAs derived from clones able to grow at the restrictive temperature were sequenced One of them encoded a protein predicted to have an N-terminal signal sequence followed by a cellulose binding domain A major part of the remaining sequence was found

to have sequence similarity with plant expansins in a BLASTdatabase search (Fig 1C) Based on its swelling activity on cotton fibers (see below) the protein was named swollenin (SWOI) and the gene swo1

The genomic copy of the swo1 cDNA was isolated from a cosmid library, subcloned and sequenced The gene contains five short introns (Fig 1A) The promoter contains a putative TATA box 90 bp and a putative binding sequence

of the glucose repressor protein CREI [32] 117 bp upstream from the translation start codon (data not shown) The putative swollenin protein starts by a typical signal sequence It is followed by two glutamic acids In the

T reesei cellulases EGI (also known as Cel7B) [33] and EGII [34] there are two glutamic acids at the N-terminus and in CBHI there is one [35], and the N-termini of these enzymes are blocked by a pyroglutamic acid residue By analogy, it is suggested that the swollenin signal sequence would be 18 amino acids in length and be cleaved before the two glutamines in the sequence (Fig 1b) The SWOI has three potential N-glycosylation sites at positions 160, 336 and 406

The swollenin cellulose binding domain (CBD) has the typical sequence features of fungal CBDs The amino acids invariant in the CBDs of the T reesei cellulases are conserved in swollenin (Fig 1B) In the NMR structure solved from the CBHI CBD there are two disulphide

bridges [36] Based on a close proximity of an additional

cysteine pair in the modelled structures of the CBHII and

EGI CBDs it has been suggested that they would have a

third disulphide bond [37] The swollenin CBD has six

cysteines at positions conserved with those of the CBHII

(also known as Cel6A) CBD and thus it probably has three

disulphide bridges as well The residues forming the flat

surface binding to cellulose are conserved in the swollenin

CBD with one exception (Fig 1B) Position 8 in the

swollenin sequence has a phenylalanine while the other

cellulases have tyrosine or tryptophan The linker region of

the swollenin is rich in serines and threonines and is

expected to be heavily O-glycosylated [38] Without protein

structure data the length of the linker cannot be determined

unambiguously However, the region rich in serines and

threonines in SWOI apears to be among the longest found

in T reesei enzymes, approximately 50 amino acids The region of SWOI between the putative linker and the expansin-like area shown in Fig 1c does not match with any sequences in databases

The C-terminal two-thirds of the swollenin show clear amino-acid similarity with plant expansins (Fig 1C) The identity between swollenin and individual a- or b-expansins

in pairwise comparisons is about 25% over an area of about

200 amino acids The alignment of the swollenin with two expansin sequences (Fig 1C) suggests that two large insertions have occurred in the swollenin gene in the N-terminal half of its expansin-like domain The identity between the a- and b-expansins is 20–25% and there are five sequence elements that are well conserved between the expansin categories [6] Four of the elements form the best conserved parts between swollenin and the expansins and

Fig 1 The basic structure of the swollenin protein (A), alignment of the swollenin CBDwith the CBDsequences of T reesei cellulases (B), Alignment

of the swollenin with two a-expansins (C) and The alignment of swollenin (SWOI) with FnIII repeat sequences (D) In (A), vertical arrows indicate the intron positions SS, signal sequence; CBD, cellulose binding domain, QQ, two glutamines suggested to form the N-terminus of the mature swollenin The numbering refers to amino-acid positions in the mature protein In (B), the invariant amino acids are boxed and amino acids forming the flat surface interacting with cellulose are shaded Lines below the sequences show the proposed disulphide bridges of the SWOI CBD (C) Alignment of the swollenin with two a-expansins, LeEx1 of tomato (5) and CuExS2 of cucumber (4) Invariant amino acids are shown by asterisks and conservative substitutions by dots The regions best conserved between a- and b-expansins are underlined in CuExS2 The conserved cysteines are shown by arrows and positions with conserved aromatic amino acids in all three sequences by + 0 The regions with homology to the FnIII repeats in titin are in bold in SWOI (D) The alignment of swollenin (SWOI) with FnIII repeat sequences of human titin and a consensus sequence

of the bacterial FnIII repeats (BACT) The amino acids strictly conserved between the bacterial and mammalian FnIII repeats are shown by asterisks.

thus they are probably functionally important The

N-terminal half of the expansins contains eight conserved

cysteines with a spacing similar to that of cysteines in the

chitin binding domain of wheat germ agglutinin [4] Seven

of these cysteines are conserved in swollenin Aromatic

amino acids are often important in the interaction of

enzymes and their carbohydrate substrates In the alignment

between swollenin and expansins there are eight positions

where an aromatic amino acid is conserved (Fig 1C)

Alignments of swollenin with individual expansins

sug-gest that it is better conserved with b-expansins than

a-expansins

There are two short sequences in swollenin that show

relatively strong conservation with fibronectin type III

(FnIII) repeats of mammalian titin proteins (Fig 1D)

Interestingly, such repeats have been found in prokaryotic

hydrolases such as cellulases, chitinases and amylases [39,40]

but thus far not in fungal enzymes The amino acids

invariant between the bacterial hydrolases and mammalian

FnIII repeats are conserved in swollenin Unlike the

continuous FnIII repeats in the bacterial enzymes, the

region with similarity to titin in swollenin is divided into two

parts about 170 amino acids apart

Regulation of the swollenin gene

The cellulase and hemicellulase genes of Trichoderma reesei

are regulated by the carbon source [12,41] and thus it was of

interest to analyze if the swollenin gene is regulated in a

similar manner The T reesei strain QM 9414 was grown in

shake flasks on different carbon sources and Northern

hybridization was performed The role of sophorose, a

strong cellulase inducer, in the swollenin gene regulation

was studied by adding it to a Trichoderma culture grown on

the neutral carbon source glycerol The swo1 mRNA level

was undetectable in the glucose culture sample in a short

exposure (Fig 2, lane 1), but in a long exposure a very low

level was observed (lane 9) In a late stage of a glucose

cultivation the swo1 gene was derepressed (lane 2) In

sorbitol (lane 3) and glycerol samples (lane 4) a low mRNA

level was present, and when sophorose was added to the

glycerol culture, strong induction of swo1 occurred (lane 5)

In media with lactose and cellobiose the swo1 mRNA level

was moderate and in a medium with cellulose it was at its

highest

Production of SWOI byT reesei Polyclonal antibodies against SWOI were obtained by immunizing rabbits with a synthetic peptide designed based

on the swollenin sequence The expected molecular mass of the deduced SWOI protein is 49 kDa, but the antibodies recognize in a Western a protein of approximately 75 kDa

in a T reesei supernatant from a cellulose-based culture (Fig 3, lanes 1 and 3) The difference between the calculated and observed molecular masses can not be explained by N-glycosylation, because endoglycosidase H that removes N-glycans changes the apparent molecular mass of SWOI only slightly (Fig 3, lane 4) Also the SWOI produced in yeast and A niger var awamori gained a molecular mass close to 75 kDa when they were treated with endoglycosi-dase H (see below) This band was also absent from the supernatants of the swo1 disruptants (Fig 3) These data show that the 75 kDa band is indeed derived from the SWOI protein The75 kDa band could not be observed in a

T reeseiculture filtrate from a culture grown on glucose (Fig 3, lane 2) Thus the very low basal expression detected

at the mRNA level (Fig 2) was undetectable in the Western analysis As estimated from Western blotting, the produc-tion level of SWOI in the T reesei culture analysed was about 1 mgÆL)1 This is by far less than the production levels

of the major cellulases

Disruption of the swo1 gene The swo1 gene was disrupted from the genome of T reesei

by replacing it with a hygromycin resistance cassette Two disruptants were shown by Southern analysis to be single-copy transformants where the gene replacement had

Fig 2 Regulation of the swo1 gene according to the carbon source.

Northern analysis of RNA samples isolated from mycelia grown in a

medium with (1) glucose (2) glucose, sample taken after glucose

depletion (3) sorbitol (4) glycerol (5) glycerol, induced by sophorose (6)

cellobiose (7) lactose (8) Solka floc cellulose (9) glucose, 7 times longer

film exposure The gpd1 probing serves as a loading control.

Fig 3 Western detection of SWOI by polyclonal antibodies from cul-ture supernatants of T reesei and yeast Lane 1, T reesei culcul-ture per-formed in a medium inducing cellulase production (1 lL of culture medium loaded); lane 2, T reesei culture performed in a medium with glucose as the carbon source (15 lL; lane 3, T reesei culture super-natant (10 lg of total protein); lane 4, T reesei culture supersuper-natant treated with endoglycosidase H (3 lg of total protein); lane 5, yeast strain expressing swo1 cDNA (the sample corresponds to 0.5 mL of supernatant); lane 6, yeast strain expressing swo1 cDNA, treated with endoglycosidase H (0.2 mL of original supernatant); lane 7, yeast strain carrying the expression vector alone (0.5 mL of original super-natant); lanes 8, 9, swo1 disruptant T reesei strains (15 lL of culture supernatant); lane 10, the parental strain of the swo1 disruptants (15 lL of culture supernatant); lane 11, Western detection of SWOI purified from A niger var awamori (100 ng); lane 12, Western detec-tion of purified SWOI, endoglycosidase H treated (30 ng); lane 13, Coomassie-stained SWOI purified from A niger var awamori (3 lg).

occurred (data not shown) Western analysis of their culture

supernatants further confirmed that they do not produce the

SWOI protein (Fig 3, lanes 8 and 9)

We attempted to demonstrate the phenotype of the swo1

disruption, its effect either on the formation of the T reesei

cell wall and growth of the fungal mycelium or on the

degradation of cellulosic carbon sources by the fungus This

was performed by comparing the growth rates of the

disruptants and the parental strain on plates having glucose

or different cellulosic compounds as carbon sources The

compounds tested were two commercial celluloses, filter

paper and a complex grain-based carbon/nitrogen source

[23] No significant differences in the growth rates could be

observed between the strains on any of the carbon sources

and thus swo1 disruption had no apparent phenotype in our

experiments

Non-stringent hybridization of T reesei genomic DNA

was performed with a swo1 gene fragment encoding the

expansin-like domain as a probe Hybridization at 48C

revealed several other bands in addition to the ones

originating from swo1, suggesting that there are other genes

having expansin-like domains present in the T reesei

genome in addition to swo1 (Fig 4) The presence of these

genes could compensate the lack of swo1 in the disruptants

and thus explain the result of the disruption experiment

Characterization of the swollenin preparations

When the swollenin cDNA was expressed in S cerevisiae

under the PGK1 promoter in a multicopy plasmid, Western

analysis of bioreactor culture supernatants showed that a

heterogeneous high molecular mass protein reacting with

the swollenin antibodies was produced by the yeast (Fig 3,

lane 5) In many instances it has been shown that yeast tends

to overglycosylate heterologous proteins, e.g the T reesei

cellulases CBHI and CBHII [42] When the swollenin

produced in yeast was treated by endoglycosidase H to remove N-glycans, it gained an apparent molecular mass close to the swollenin produced by Trichoderma (Fig 3, lane 6) The production level of SWOI in yeast was approxi-mately 25 lgÆL)1 as estimated from Western blotting experiments

Swollenin was also produced in A niger var awamori and after a single step purification procedure the purified swollenin protein was obtained for biochemical character-ization The SWOI expressed in this host migrated as two relatively diffuse bands with apparent molecular masses between 80 and 95 kDa (Fig 3, lanes 11 and 13), and

Endo-H treatment reduced the molecular mass to the same level as SWOI produced by T reesei (Fig 3, lane 12) Thus A niger var awamori slightly overglycosylated SWOI Activities of purified swollenin against hydroxyethyl cellulose (HEC), b-glucan, xylan and mannan were measured and the results are shown in Table 1 Minor hydrolytic activity on b-glucan, xylan and mannan, but not on HEC, was observed for the purified swollenin protein expressed in A niger

Demonstration of the swollenin activity on solid substrates

The swollenin expressed in yeast The activity of the swollenin produced in yeast towards cellulosic materials was shown by treatments of cotton fibers and filter paper Cotton fibers were incubated with concentrated yeast supernatants from bioreactor cultivations of the SWOI-producing yeast (approximately 0.125 lgÆmL)1of SWOI) and the control strain with vector alone or, as controls, with the T reesei cellulases EGII, CBHI (5 lgÆml)1) and the cellulose binding domain of CBHI After the treatment the fibers were removed from the reaction mixture by filtering, rinsed, sonicated with glass beads, stained and analyzed by light microscopy Soluble reducing sugars were measured from the reaction mixture Treatment with the control yeast supernatant did not change the fiber structure (Fig 5A) The supernatant of the yeast strain producing swollenin caused local disruption of the fiber structure that became visible only after sonication This was seen as swollen areas occurring along the fibers (Fig 5B) CBH I caused some light fibrillation of the fibers (Fig 5C), whereas the treatment with EG II resulted in damaged and rugged outlook of the fibers accompanied by fiber cutting (Fig 5E) Din and coworkers [43] have reported on disruption of cellulosic fibers by a bacterial cellulose binding domain No modification of fiber surface could be detected in our experiment with the fungal (CBHI) CBD by the light microscopical method used (Fig 5D), suggesting that the effect of SWOI on the fibers is not caused by its CBD The treatment of the cotton fibers with the yeast supernatants or

Table 1 Characteristics of the swollenin preparation purified from

A niger var awamori.

Protein (mgÆmL)1)a

Activity (nkatÆmL)1) HEC b-glucanase Xylanase Mannanase

a Lowry protein.

Fig 4 Southern hybridization of T reesei genomic DNA with the

region encoding the expansin-like domain from the swo1 gene, performed

at stringent (68 °C) and nonstringent (48 °C) conditions The restriction

enzymes used are indicated.

with the CBD did not release detectable amounts of

reducing sugars In contrast, the filtrates from the

CBHI-and EGII-treated fibers contained 0.08% CBHI-and 1.61%

reducing sugars of the original dry mass, respectively

To test the effect of swollenin on paper, filter paper strips were incubated in concentrated culture supernatants of the swollenin-producing and control yeast strains and measured for their wet tensile strength The data shows how much load each strip of paper could hold before it broke; breakage

at a lower mass indicates less tensile strength (Table 2) The average load is only slightly decreased when broth from yeast which does not contain the swollenin gene is used However, the same amount of broth from the yeast expressing the swollenin gene results in a 15–20% decrease

in the average load compared to the control broth Incubation in 8M urea decreases the average load the paper can bear by about 40% compared to buffer alone The purified swollenin Fragments of Valonia cell walls were used as the solid substrate for studies on the mode of action of the purified swollenin This algal cell wall is made

of highly crystalline cellulose with a layered structure as shown in Fig 6A Fragments of the cell wall were incubated individually with the purified cellulases, CBH I and EG II, and swollenin and alterations in the cell wall structure were followed microscopically The cellulases modified the cell wall fragments with a concomitant release of soluble sugars (Table 3) Treatment with EG II disrupted totally the cell wall structure resulting in a milky solution whereas with CBH I disintegration of cell wall to fibrils was observed (Fig 6) The action of swollenin resembled that of CBH I, but integrity of the cell wall was partially retained and no soluble sugars were released

D I S C U S S I O N

T reeseiproduces one of the most powerful mixtures of extracellular enzymes for efficient hydrolysis of the plant polysaccharides cellulose and hemicellulose This fungus has served as a model, and extensive studies on the biochem-istry, genetics, regulation, structure-function relationships and applications of T reesei enzymes have been carried out [44] The discovery of the expansin-like protein SWOI in

Table 2 The average peak load a strip of filter paper could bear before breakage The filter paper strips were treated with buffer, yeast culture supernatants or urea as indicated The results are the average of 3 or 4 readings.

Sample Average Peak Load (g) SD

Swollenin-producing yeast 186 5.0

Fig 5 Light microscopy of cotton fibers treated with culture super-natant of control yeast with the vector alone (A), supersuper-natant of swollenin-producing yeast (approximately 0.125 lgÆL)1of SWOI) (B), and isolated T reesei cellulases CBHI (C) and EGII (E) and the cellulose binding domain of CBHI (D) (5 lgÆmL)1) The swollen areas caused by SWOI treatment are shown by arrows Small fibrils can be seen on the surface of fibers treated by CBHI EGII caused modification of the fibers that can be seen as rugged surface outlook.

T reeseiprovides new insight to the mechanism of

micro-bial lignocellulose degradation, together with the report on

an endoglucanase with an expansin-like domain in a

pathogenic bacterium [13] and the discovery of sequence

similarity of expansins with family 45 glycosyl hydrolases

(see below)

Similarly to plant expansins, filter paper was shown to be

weakened by SWOI in an extensometer assay We also

demonstrate that the structure of mercerized cotton fibers

was changed upon swollenin treatment in a manner clearly

visible by light microscopy It can be assumed that the

swollen areas of the cotton fibers appearing after swollenin treatment coincide with the tilt/twist areas of cotton fibers, where the structure of cellulose is less ordered and more accessible for modification than in crystalline regions [45] Both cotton and filter paper consist of relatively pure cellulose, and therefore swollenin would appear to be able to open the crosslinking of cellulose fibers No reducing sugar formation was detected in the cotton swelling test, which is

in accordance with the plant expansin results published Disruption activity that was detected upon treatment of Valonia cell wall frgaments with purified SWOI is in line with the results obtained with yeast supernatants containing SWOI Although the Valonia cell wall are not representative

of the higher plant cell walls, the ability of SWOI to disrupt the Valonia cell wall without producing reducing sugars is of special interest Activity of this type has been reported for the expansins The SWOI preparate purified from A niger var awamori had a slight activity towards b-glucan, mannan and xylan, but no activity towards hydroxyethyl cellulose The detected enzyme activities were very low, e.g the specific activity of the T reesei endoglucanases EGI or EGII against b-glucan are thousands of nkatÆmg)1, whereas the SWOI preparate had an activity of 79 nkatÆmg)1 At present we can not be sure whether the activities observed in the SWOI preparation are due to trace amounts of contaminating A niger var awamori enzyme(s) or to a weak hydrolytic activity of the SWOI protein itself However, the disruption ability of solid substrate structures observed in this work is most probably not due to hydrolytic activity of SWOI, as no reducing sugar release from the solid substrates was detected in these activity tests

It has been reported that expansins have limited sequence similarity with the family 45 of glycosyl hydrolases, which includes the T reesei endoglucanase EGV [3,14,46] This similarity is in the same range in identity percentages as the similarity in an alignment between swollenin and individual expansins, but it is limited to a smaller area (data not shown) The sequence conservation between EGV and SWOI is hardly detectable and thus it is weaker than conservation between EGV and expansins The sequence motif HFD forming a part of the active site of the family 45 hydrolases [47] is conserved in the expansins, and according

to the alignment in Fig 1C would appear to be replaced by HLD in SWOI The degree of conservation between swollenin and the expansin-like domain of celA from Clavibacter michiganensis is lower than conservation between swollenin and plant expansins (data not shown)

In a recent report the b-expansin of Phleum pratense was shown to have proteinase activity and to have limited sequence similarity to papain-type proteinases [48] The authors proposed that expansins loosen the plant cell wall structure by cleaving cell wall proteins that crosslink cellulose fibers together rather than by disrupting hydrogen bonding between fibers The regions around the three active site residues of papain were suggested to be conserved in both a- and b-expansins Some amino-acid similarity between papain and swollenin can be detected at one of these regions (around Cys256 of swollenin) but the others are not conserved

An interesting feature of the T reesei swollenin is that it has a modular structure typical of fungal cellulases and some hemicellulases SWOI has an N-terminal cellulose binding domain (CBD) that is very well conserved with

Fig 6 Atomic force microscopy image of the structure of the Valonia

cell wall (A), and light microscopy of Valonia cell wall fragments after

treatment with buffer alone (B), SWOI (C, 10 lgÆmg)1), CBHI (D,

100 lgÆmg)1) and EGII (E, 100 lgÆmg)1).

Table 3 Disintegration of Valonia cell walls by the purified swollenin

and T reesei cellulases The treatments were performed at a

consis-tency of 0.25%, at + 25 C for 48 h.

Treatment

Dosage

(lgÆg)1) Effects on cell wall

Solubilized sugars (% of dw)

Swollenin 10 Partial disintegration

to fibrils

0 CBH I 10 Total disintegration

to fibrils

0.09

EG II 10 Total disintegration

to milky solution

9.2

other fungal CBDs Thus it can be expected that its function

is to bind the SWOI protein to cellulosic compounds An

other interesting feature, although much less clear in its

functional importance, is the sequence similarity to the

fibronectin III (FnIII) type repeats of mammalian titin

proteins (Fig 1D) The FnIII repeats of titin form b

sand-wich domains that have been suggested to be able to unfold

and refold easily [49] and this would make the protein able

to stretch The ability to stretch might be important for

swollenin, if its function is to allow slippage of cellulose

microfibrils in plant cell walls as suggested for expansins

Our results suggest that swollenin is a component of the

enzyme mixture produced by the fungus which is needed for

degradation of plant biomass and not, e.g in modifying the

Trichodermacell wall during the growth of the fungus The

regulation pattern of the T reesei swo1 gene is highly

reminiscent to that of the cellulase genes of this fungus [41]

The gene is induced for instance by plant materials and

certain oligosaccharides The swo1 gene has a low

expres-sion level on glucose, sorbitol and glycerol unlike the more

tightly repressed major cellulases [41] This could imply to

the interesting possibility that swollenin would be among

the enzymes that, before the onset of massive cellulose

degradation, aid in liberating a soluble inducer when the

fungus is encountering the insoluble cellulosic substrate

This soluble inducer would further induce the main

cellulolytic machinery

According to early theories on cellulose degradation, the

cellulase system of fungi like T reesei would comprise two

kinds of activities It was suggested that C1 (swelling

factor), a nonhydrolytic component would be needed to

make the substrate more accessible to Cx, the hydrolytic

component consisting of the endo- and exo-acting enzymes

and b-glucosidases that degrade the substrate to glucose

[50] A large number of hydrolytic enzymes have been

characterized but so far the C1 factor has remained

unsolved The Trichoderma C1 has not been well

charac-terized, but based on gel filtration it has been reported to

have a molecular mass of 61 kDa [51], not far from 75 kDa

that was estimated by SDS gels to be the molecular mass of

SWO1 Based on the properties of swollenin shown in this

work, it provides a possible candidate for a component of

C1 Our results also point towards the existence of other

proteins with sequence similarity to SWOI in T reesei

(Fig 4) Thus it is possible that there exist several

swollenin-like activities as is the case with the hydrolytic enzymes,

which vary somewhat in their modes of action but all

contribute synergistically to the efficient hydrolysis of the

plant polysaccharides

A C K N O W L E D G E M E N T S

We wish to thank Riitta Nurmi and Kati Uotila for excellent technical

assistance The work was supported by the Finnish National

Technology Agency (Tekes).

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