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Molecular Plant Advance Access originally published online on August 20, 2009
Molecular Plant 2009 2(5):904-909; doi:10.1093/mp/ssp060
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© The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

Loosening Xyloglucan Accelerates the Enzymatic Degradation of Cellulose in Wood

Rumi Kaidaa, Tomomi Kakua, Kei'ichi Babaa, Masafumi Oyadomaria, Takashi Watanabea, Koji Nishidab, Toshiji Kanayab, Ziv Shanic, Oded Shoseyovc and Takahisa Hayashia,d,1

a Kyoto University, RISH, Gokasho, Uji, Kyoto 611-0011, Japan
b Kyoto University, Institute for Chemical Research, Gokasho, Uji, Kyoto 611-0011, Japan
c CBD Technologies, Rehovot 76100, Israel
d Institute of Sustainability Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

1 To whom correspondence should be addressed at address a. E-mail taka{at}rish.kyoto-u.ac.jp, fax and tel. +81 774 38 3618.


   Abstract
TOP
 Abstract
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 SUPPLEMENTARY DATA
 FUNDING
 
In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

Key Words: Overexpression of xyloglucanase • saccharification • transgenic poplar • xylem

Received for publication April 5, 2009. Accepted for publication July 10, 2009.


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 SUPPLEMENTARY DATA
 FUNDING
 
1,4-β-glucans have a strong tendency to self-associate into microfibrils consisting of repeated crystalline and non-crystalline parts, each of which might be quite short (10–100 glucosyl residues) compared to the length of a whole cellulose microfibril. In plants, these microfibrils consist of many paracrystal 1,4-β-glucans, which form nanofibers 3–4 nm in width and thickness, in which surface glucans may be irregularly intercalated with hemicelluloses at various levels (O'Sullivan, 1997). The nanofibers associate to form a bundle of compact lattices between hydrophobic and hydrogen bonds that can result in Iβ crystalline regions (Hackney et al., 1994), and multiple intercalation of some paracrystal glucans can result in non-crystalline regions. Primary and secondary walls may have similar levels of intercalation, but an increased amount of cellulose occurs along with lignin deposition in the secondary wall. It is now necessary to achieve a transformational modification of wood that will allow us to use it not only as a material, but also as a source of glucose. This communication describes the saccharification of wood and how it could be modified into a source of glucose.

Cellulase does not easily hydrolyze 1,4-β-glucan intercalated with hemicellulose, nor does hemicellulase efficiently attack hemicellulose when the latter is intercalated tightly into microfibrils in wood, because plant cell elongation and expansion further tighten and fix the intercalation between 1,4-β-glucans and hemicellulose during growth. Lignin is bound to xylan and glucomannan (Imamura et al., 1994; Lawoko et al., 2006), whose constitutive degradation might also reduce lignin deposition in the walls because lignin is known to be a recalcitrant compound in cellulose hydrolysis (Chen and Dixon, 2007). The question is whether the hydrolysis of cellulose in wood can be accelerated by constitutively degrading hemicellulose during growth and thus preventing its intercalation. This hypothesis draws support from the observation that, in barley straws, xyloglucanase activity improved the total hydrolysis of lignocelluloses (Benko et al., 2008) and also by the presence of xyloglucan in secondary walls (Bourquin et al., 2002; Nishikubo et al., 2007; Mellerowicz et al., 2008; Baba et al., 2009). It should be noted that enzymatic hydrolysis is much more environmentally friendly than other processes such as acid hydrolysis. Our aim was to assess hemicellulose as a recalcitrance to the enzymatic saccharification of xylem cellulose.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 SUPPLEMENTARY DATA
 FUNDING
 
Saccharification of Xylem Powder
We produced several transgenic poplars, each of which overexpressed either xyloglucanase, cellulase, xylanase, or galactanase (plus arabinofuranosidase), and confirmed the specific degradation of each overexpressed enzyme's target polysaccharide with chemical analysis of the xylem followed by methylation analysis of hemicellulose (alkali-soluble polysaccharides) from the secondary xylem (Baba et al., 2009). Degradation occurred at the apoplastic space because the glycanase cDNAs were expressed using a signal sequence fused with a constitutive promoter and an enhancer (Park et al., 2004). Although no significant difference in lignin content was found between the transgenic lines and the wild-type (Baba et al., in press at Molecular Plant), the partially methylated alditol acetates of the hemicelluloses showed that 4-linked xylose was the most predominant derivative in the wild-type, indicating that xylan is a major component of hemicellulose in poplar stems.

Consistently throughout the incubation period, more enzymatic saccharification of xylem powder took place in the transgenic poplars overexpressing xyloglucanase, xylanase, and cellulase than in the wild-type poplars (Figure 1). More cellulose hydrolysis took place in xylem overexpressing xyloglucanase than in xylem overexpressing xylanase or cellulase. At 48 h after the start of incubation, up to 57% of cellulose was hydrolyzed in the xylem overexpressing xyloglucanase, up to 52% of cellulose was hydrolyzed in the xylem overexpressing xylanase, and up to 46% of cellulose was hydrolyzed in the xylem overexpressing cellulase. The latter data point corresponds to the enhanced conversion into glucose seen in rice overexpressing cellulase (Oraby et al., 2007). In the wild-type xylem, meanwhile, at most, 31% of cellulose was hydrolyzed in 48 h. In contrast, xylan was hydrolyzed at approximately the same rate in all transgenic xylems and the wild-type xylem; it was almost completely hydrolyzed in all xylems at 48 h. This is in agreement with the observation by O'Dwyer et al. (2008) that xylan hydrolysis was independent of cellulose digestion during the initial hydrolysis stage of poplar wood samples. The results suggest that xyloglucan, xylan, and glucomannan could be intercalated into cellulose microfibrils, in which the degradation of xyloglucan has been demonstrated to occur with greater efficiency than the enzymatic hydrolysis of cellulose does.


Figure 1
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  		Figure 1. Compositions of Monosaccharides Released by Enzymic Hydrolysis of Transgenic Xylem during the Time Course of the Study.

The amount of each sugar released was calculated from the reducing sugar (Supplemental Table 1) and its alditol acetates. The values for the composition of the acetates represent the mean of independent lines for each transgenic line, with individual values varying from the mean by 2.5%. Wt, wild-type; XEG, transgenic (xyloglucanase); XYL, transgenic (xylanase); CEL, transgenic (cellulase); GAL, transgenic (galactanase).

 
X-ray diffraction analysis revealed that enzymatic digestion of xylem powder occurred between the crystalline and non-crystalline natures in a pattern similar to that of the undigested xylem preparation, due to two broad equatorial diffractions centered at ~15.5° and ~22° (Supplemental Figure 1), although the level of crystallinity and the crystal size of the microfibrils was slightly decreased in the digested residual xylem overexpressing xyloglucanase (Table 1). The high rate at which cellulose microfibrils at the crystalline region in the xylem overexpressing xyloglucanase degraded suggests that the surface of crystalline cellulose might be highly intercalated with xyloglucan.


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  		Tabel 1. Potential microfibril crystal size and crystallinity. The individual values represent the mean of three different lines for each xylem varying from the mean by < 0.8%.

 
Saccharification of Xylem Section
Scanning electron microscopy revealed that, in the first 48 h of incubation, enzymatic saccharification occurred in all types of cell walls at all loci, meaning that cellulases and hemicellulases in the enzyme preparation attacked all types of cells between the inner and outer surfaces of walls in each transverse section of xylem (Figure 2A and 2B). This saccharification eventually led to the separation of the inner and outer walls through the removal of the lamella between them, in a process that might result in a thinner wall and less elasticity in the xylem overexpressing xyloglucanase than in the wild-type xylem (Figure 2C–2F).


Figure 2
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  		Figure 2. Scanning Electron Microscopy of Transversely Sliced Xylems from the Wild-Type and the Transgenic Poplar Overexpressing Xyloglucanase.

(A) Wild-type whole xylem after 48-h enzymatic hydrolysis.

(B) Transgenic whole xylem after 48-h enzymatic hydrolysis.

(C) Wild-type xylem before enzymic hydrolysis.

(D) The white square in (A) is shown as magnified view.

(E) Transgenic xylem before enzymic hydrolysis.

(F) The white square in (B) is shown as magnified view. Bars = 500 µm for panels (A) and (B) and 10 µm for panels (C)–(F).

 
Simultaneous Enzymic Saccharification and Fermentation
When saccharification was accompanied by fermentation with yeast, ethanol production was higher in the enzymatic hydrolysate of the xylem overexpressing xyloglucanase than in any other xylem sample (Figure 3). It was higher in the enzymatic hydrolysate of the xylems overexpressing xylanase and cellulase than in that of the wild-type xylem, and slightly lower in that of the xylem overexpressing galactanase. The pattern of relative ethanol production among the transgenic xylems was similar to the pattern of relative cellulose degradation (Figure 1). The levels of ethanol production were theoretically 10–15% higher than those of fermentable sugars (glucose, mannose, and galactose) produced during saccharification. At 48 h into this process, the level of cellulose hydrolysis was calculated to be 65% for the xylem overexpressing xyloglucanase and 35% for the wild-type xylem.


Figure 3
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  		Figure 3. Ethanol Production Accompanying Enzymic Hydrolysis of Xylem.

The values are from Supplemental Table 1. Wt, wild-type; XEG, transgenic (xyloglucanase); XYL, transgenic (xylanase); CEL, transgenic (cellulase); GAL, transgenic (galactanase). The values of the ethanol production represent the mean of independent lines for each transgenic line, with individual values varying from the mean by 5.5%.

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 SUPPLEMENTARY DATA
 FUNDING
 
The various transgenic effects all support the idea that the loosening of xyloglucan could accelerate the saccharification of cellulose microfibrils (Figure 1). Certain overexpressions could cause microfibril relaxation resulting from the cleavage of cross-linked xyloglucans, which could accelerate not only cellulose biosynthesis in vivo during growth (Park et al., 2004), but also cellulose degradation in vitro in the xylem (Figure 1). The level of xyloglucanase activity (6400 units used for 100 mg xylem) in the cellulase preparation was about 120 times that in the xylem (50 units/100 mg of living xylem) overexpressing xyloglucanase. Thus, the expression of xyloglucanase in the stem constitutively was more effective than the treatment of the xylem with xyloglucanase preparations, even though the preparations included cellulase and other hemicellulases working together to degrade the xylem. Although the overexpression of plant cellulase can also loosen xyloglucan intercalation, leading to a decrease in paracrystalline sites of cellulose microfibrils (Shani et al., 2004; Park et al., 2003; Hartati et al., 2008), the intercalation was more effectively removed by the overexpression of xyloglucanase, leading to a decrease in intercalation and a relative increase in the number of crystalline regions (Table 1). In addition, the transgenic poplars overexpressing xyloglucanases are now under their field trial in Japan (Supplemental Figure 2). By using 3-year-old woods, the levels of cellulose degradation and ethanol production were still markedly greater in the xylem overexpressing xyloglucanase than in the xylem of the wild-type plant.

Decreases in xylan and glucomannan content did not reduce lignin deposition in the xylems overexpressing xylanase and cellulase, although an overexpression of these enzymes would be expected to decrease the lignin content. Nevertheless, the complete hydrolysis of xylan did not increase the level of cellulose hydrolysis. Although hemicelluloses in pulp facilitate nanofibrillation and improve the physical properties of nanocomposites (Iwamoto et al., 2008), it has been confirmed that xyloglucan acts as a structural inhibitor for cellulase in the saccharification of xylem.

There might have been some product inhibition during saccharification rather than simultaneous enzymic saccharification and fermentation, because the levels of ethanol production were theoretically 10–15% higher than those of fermentable sugars (glucose, mannose, and galactose) produced during saccharification (Figures 1 and 3). The higher the level of saccharification rose, the more inhibition occurred. Therefore, the inhibition was rather higher for the xylem overexpressing xyloglucanase. It should be noted that although ethanol fermentation was reported to be inhibited by 5-hydroxymethyl furfural, furfural, and vanillin formed in acid hydrolysis or thermal pretreatment (Miyafuji et al., 2005), no corresponding inhibitory compounds were found in the enzymic hydrolysate.

Our results show that xyloglucan serves as a key hemicellulose and a tightening tether of cellulose microfibrils in the secondary walls. If the tether could be loosened rather than tightened during growth, not only would the trees grow faster (Park et al., 2004; Shani et al., 2004; Hartati et al., 2008), but the cellulose microfibrils could be hydrolyzed by cellulase at a high rate (Figure 1). These predictions are in agreement with the finding (Nishikubo et al., 2007; Baba et al., in press at Molecular Plant) that xyloglucan tightens gelatinous layers to the S2 layer in the secondary walls and provides tension to the wall structure. Therefore, the genetic reduction of xyloglucan in xylem has been confirmed to accelerate the hydrolysis of cellulose microfibrils in wood. Such technology could be applied either as in fibril rather than in wall modification, such as reduction of lignin, or as in planta modification, such as autohydrolysis during post-harvest. All of these modifications, one after another, will be necessary to facilitate bioprocess consolidation for bioethanol production.


   METHODS
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
SUPPLEMENTARY DATA
 FUNDING
 
Transgenic Poplars
We used 14 clones of transgenic poplars overexpressing Aspergillus xyloglucanase (AaXEG2, AY160774 [GenBank] ) with the PaPopCel1 (D32166 [GenBank] ) signal peptide, eight clones of transgenic poplars overexpressing xylanase (HvXYL1, U59312 [GenBank] ), four clones of transgenic poplars overexpressing Arabidopsis cellulase (AtCel1, X98544 [GenBank] ) with its signal peptide, and eight clones of transgenic poplars overexpressing galactanase (Tv6GAL, AB104894 [GenBank] ) including arabinofuranosidase (RsAraf1, AB234292 [GenBank] ) with Arabidopsis arabinogalactan protein signal (AtAGP4, AF082301 [GenBank] ) (Baba et al., 2009).

Xylem Preparation
The poplars used are derived from cut plants propagated many times for 4 years. One-month-old transgenic poplar stems (about 30 cm in length) were harvested, their barks were peeled and their 11th to 20th internodes (xylem) were dried in an oven at 70°C. The first internode was defined as the one below the uppermost leaf of 0.5-cm length. The xylem was then cut into pieces and milled to a powder using a ball mill at a speed of 15 rps for 10 min for saccharification either alone or together with fermentation.

Enzymatic Hydrolysis
One hundred mg of xylem preparation was impregnated with water, autoclaved at 120°C for 3 min, and washed once with water by centrifugation. A commercial cellulase preparation (Meicelase, Meiji Seika Co., Tokyo, Japan) derived from Trichoderma viride was used for the digestion of xylem. The enzyme preparation contained endocellulases, exocellulases (CBHI and CBHII), xyloglucanase, xylanase, galactanase, and polygalacturonase. Hydrolysis of hemicellulose (24% KOH-soluble fraction) with the enzyme preparation resulted in monosaccharides. Enzymatic hydrolysis of the xylem preparation was performed in 2 ml of 50 mM sodium acetate buffer, pH 4.8, containing 0.02% Tween 20 and 0.4 filter paper units of a cellulase preparation (2.0 mg). One filter paper unit is defined as 1 µmol of glucose released per minute from 50 mg of Whatman 3MM filter paper at 45°C in a rotary shaker set at 135 rpm. About 100 µl of the supernatant was collected at 6, 24, and 48 h after the start of hydrolysis and used for sugar analysis. The sugar released was estimated as reducing sugar by the Nelson-Somogyi method (Somogyi, 1952). Furthermore, free sugars released were directly analyzed as their alditol acetates using gas chromatography as described above (Hayashi, 1989). Two mg cellulase preparation contained about 6400 units of xyloglucanase activity, which was assayed viscometrically at 35°C for 2 h with 0.1 ml of enzyme preparation plus 0.9 ml of 10 mM sodium phosphate buffer (pH 6.2) containing 0.65% (w/v) tamarind xyloglucan in Cannon semimicroviscometers (Cannon Instrument Co., State College, PA, USA). One unit of xyloglucanase activity is defined as the amount of enzyme required to cause 0.1% loss in viscosity in 1 min under such conditions (Park et al., 2004).

X-Ray Diffraction
The xylem preparation was subjected to X-ray diffraction measurement using a Rigaku (Tokyo, Japan) RINT 2000 with Kβ-filtered CuK{alpha} radiation at 40 kV and 50 mA. The degree of crystallinity of native cellulose was calculated from the X-ray diffraction patterns according to the method described by Segal et al. (1959). The line broadening and crystal size of cellulose microfibrils were calculated from the X-ray diffraction patterns according to Scherrer's equation.

Microscopy
The dried xylem was dipped in water under reduced pressure and autoclaved at 120°C for 2 min. The wet xylem was cut by hand with a razor into transverse sections approximately 0.5–1.0 mm wide. Each section was digested with the cellulase preparation used for saccharification, then washed three times with water and dried at 40°C overnight. Each section was then observed under a field emission scanning electron microscope (FE–SEM).

Ethanol Production
Simultaneous enzymic saccharification and fermentation were induced in a mixture containing the xylem preparation in 2 ml of 50 mM sodium acetate buffer, pH 4.8, 0.02% Tween, 0.4 filter paper units of a cellulase preparation, and a seed culture of Saccharomyces cerevisiae (SH1089) with yeast nutrients (4 mg (NH4)2HPO4, 0.2 mg MgSO4/7H2O, and 8 mg yeast extract). This mixture was incubated at 45°C in a rotary shaker set at 135 rpm. About 100 µl of the supernatant was collected at 6, 12, 24, and 48 h after the start of hydrolysis and used for ethanol analysis. The ethanol formed was measured by gas chromatography on a Supelcowax-10 (Supelco, St Louis, MO, USA) column (0.53 mm i.d. x 15 m) at 50°C using an Agilent (Santa Clara, CA, USA) gas chromatograph. Isopropanol was used as an internal standard.


   SUPPLEMENTARY DATA
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 SUPPLEMENTARY DATA
FUNDING
 
Supplementary Data are available at Molecular Plant Online.


   FUNDING
TOP
 Abstract
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 SUPPLEMENTARY DATA
 FUNDING
 
Funding for this work was provided by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and by JSPS KAKENHI (grant nos 19208016 and 19405030). This work is also part of the outcome of the JSPS Global COE Program (E-04): In Search of Sustainable Humanosphere in Asia and Africa. No conflict of interest declared.

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