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  • Multidimensional NMR analysis reveals truncated lignin structures in wood decayed by the brown rot basidiomycete Postia placentaemi_2417 1091..1100

    Daniel J. Yelle,1† Dongsheng Wei,1,2† John Ralph3

    and Kenneth E. Hammel1,4* 1USDA Forest Products Laboratory, Madison, WI 53726, USA. 2Department of Microbiology, Nankai University, Tianjin 300071, China. 3Department of Biochemistry and DOE Great Lakes Bioenergy Research Center and 4Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA.

    Summary

    Lignocellulose biodegradation, an essential step in terrestrial carbon cycling, generally involves removal of the recalcitrant lignin barrier that other- wise prevents infiltration by microbial polysaccha- ride hydrolases. However, fungi that cause brown rot of wood, a major route for biomass recycling in coniferous forests, utilize wood polysaccharides efficiently while removing little of the lignin. The mechanism by which these basidiomycetes breach the lignin remains unclear. We used recently devel- oped methods for solubilization and multidimen- sional 1H–13C solution-state NMR spectroscopy of ball-milled lignocellulose to analyse aspen wood degraded by Postia placenta. The results showed that decay decreased the content of the principal arylglycerol-b-aryl ether interunit linkage in the lignin by more than half, while increasing the fre- quency of several truncated lignin structures roughly fourfold over the level found in sound aspen. These new end-groups, consisting of benz- aldehydes, benzoic acids and phenylglycerols, accounted for 6–7% of all original lignin subunits. Our results provide evidence that brown rot by P. placenta results in significant ligninolysis, which might enable infiltration of the wood by polysaccha- ride hydrolases even though the partially degraded lignin remains in situ. Recent work has revealed that

    the P. placenta genome encodes no ligninolytic peroxidases, but has also shown that this fungus produces an extracellular Fenton system. It is accordingly likely that P. placenta employs electro- philic reactive oxygen species such as hydroxyl radicals to disrupt lignin in wood.

    Introduction

    Most terrestrial biomass is sequestered in lignocellulose, the principal structural material of vascular plants. Accord- ingly, the continuous biodegradation of this complex natural composite is a key step in the global carbon cycle (Zeikus, 1981). However, few organisms can metabolize lignocellulose because its cellulose and hemicelluloses, the components that can support microbial growth, are embedded in lignin. The lignin prevents saccharolytic enzymes from accessing the underlying polysaccharides, and is recalcitrant to hydrolytic depolymerization because it is a combinatorial, racemic polymer of phenylpropanoid subunits connected via ether and carbon–carbon bonds (Ralph et al., 2004a). Hence, any organism that depends on lignocellulose as a source of carbon and energy must have some mechanism to penetrate or disrupt this lignin barrier.

    Wood decay basidiomycetes are the most efficient bio- degraders of lignocellulose. Most of these, termed white rot fungi, cleave the encrusting lignin oxidatively to access the polysaccharides they require for growth, and are generally thought to employ several specialized per- oxidases to promote ligninolysis (Martínez, 2002). However, brown rot basidiomycetes, a phylogenetically related group principally responsible for lignocellulose recycling in coniferous forests (McFee and Stone, 1966), utilize wood polysaccharides efficiently while leaving most of the lignin in situ (Cowling, 1961). They also gen- erally lack detectable ligninolytic peroxidase activity (Eriksson et al., 1990).

    The question of how brown rot fungi access wood polysaccharides without removing the lignin remains per- plexing. Part of the explanation is probably that they produce extracellular Fe2+ and H2O2, which are small enough to infiltrate lignocellulose and react with each other within the wood cell wall via the Fenton reaction

    Received 19 May, 2010; accepted 3 December, 2010. *For corre- spondence. E-mail [email protected]; Tel. (+1) 608 2319528; Fax (+1) 608 2319262. †These authors contributed equally to this work.

    Environmental Microbiology (2011) 13(4), 1091–1100 doi:10.1111/j.1462-2920.2010.02417.x

    Published 2011. This article is a US Government work and is in the public domain in the USA.

    mailto:[email protected]

  • (H2O2 + Fe2+ + H+ → H2O + Fe3+ + •OH) to produce hydroxyl radicals (•OH) that oxidatively cleave cellulose and hemicelluloses. Many brown rot basidiomycetes produce extracellular hydroquinones (Goodell et al., 1997; Hammel et al., 2002), which reduce Fe3+ and O2 to drive Fenton chemistry (Guillén et al., 1997; Kerem et al., 1999; Gómez-Toribio et al., 2009). Brown rot fungi have also been reported to oxidize a variety of polymers, including cellulose, to products that suggest the involve- ment of •OH (Kirk et al., 1991; Kerem et al., 1998; Cohen et al., 2002).

    However, brown rot fungi also produce cellulases and hemicellulases (Eriksson et al., 1990), which suggests a requirement for ligninolysis to enable cell wall infiltra- tion by these enzymes. In agreement, there are some indications that lignin is cleaved during brown rot: (i) the fungi cause low to moderate decreases in the acid- insoluble material (Klason lignin) that is generally interpreted as residual lignin after decay (Eriksson et al., 1990), (ii) ultrastructural changes characteristic of ligninolysis have been observed in the secondary cell wall of decayed wood specimens (Eriksson et al., 1990), (iii) a solid-state 13C NMR spectrum of brown-rotted wood was reported to exhibit a signal attributable to lignin benzoic acid residues (Davis et al., 1994) and (iv) it has been observed that brown-rotted lignin is enriched in carbonyl and carboxyl groups (Kirk, 1975) while being depleted in major intermonomer linkages (Yelle et al., 2008a; Martínez et al., 2011). However, structures that are strongly diagnostic for ligninolysis have not yet been identified or quantified in brown-rotted wood.

    The observation of cleaved lignin structures in biode- graded wood is technically challenging. Solvent extraction of lignins from lignocellulose is never quantitative, and thus results in sample fractionation that may obscure or magnify differences between degraded specimens and controls upon subsequent analysis. Solid-state NMR spectroscopy addresses the fractionation problem, but yields broad, overlapping signals that make it difficult to pinpoint structures diagnostic for ligninolysis. Recently, new methods for the solubilization of entire lignocellulose samples have addressed some of these problems by

    making it possible to exploit the enhanced signal disper- sion provided by multidimensional solution-state NMR spectroscopy (Lu and Ralph, 2003; Ralph et al., 2006; Yelle et al., 2008a,b; Hedenström et al., 2009; Kim and Ralph, 2010). The principal caveat is that the samples must first be ball-milled, but research to date indicates that this procedure causes only minor increases in detect- able cleaved lignin structures (Ikeda et al., 2002; Kim and Ralph, 2010). Here we have taken this approach to char- acterize changes that occur in lignin after wood decay by the brown rot fungus Postia placenta.

    Results

    Initial processing and analysis

    We grew P. placenta on aspen wood, and after 16 weeks of decay we pooled 32 specimens exhibiting a dry weight loss of 69.7 � 5.4%. The chemical composition of the biodegraded wood was typical of decay caused by this fungus (Table 1) (Eriksson et al., 1990). After grinding this material coarsely, we ball-milled 1 g of the resulting wood meal for 12 h at 300 r.p.m. For comparison, we pooled and ground 32 wafers of sound aspen sapwood, and then ball-milled 1 g of this material for 12 h at 600 r.p.m.

    We then determined the methoxyl contents of the degraded and control samples, using an iodometric method that is quantitative with solid lignocellulosic specimens (Chen, 1992), so we could employ the lignin methoxyl NMR signal in each sample as an internal stan- dard for quantification of other lignin structures. A loss of lignin methoxyls is typical after brown rot (Kirk and Adler, 1970; Kirk, 1975), and in this case their content declined from 1.82 to 1.00 mmol per g original wood. We next acetylated a portion of each sample to make it soluble in perdeuterated dimethylsulphoxide (DMSO-d6) for solution-state NMR experiments (Lu and Ralph, 2003; Yelle et al., 2008a). In addition, we treated some of each ball-milled preparation with a cellulase mixture to remove most of the polysaccharides, which made the samples soluble in DMSO-d6 without acetylation (Ralph et al., 2006).

    Table 1. Chemical composition of sound and decayed aspen wood samples.

    Sample Klason lignin (mg)

    ASL (mg)

    Glucose (mg)

    Xylose (mg)

    Mannose (mg)

    Galactose (mg)

    Arabinose (mg)

    Rhamnose (mg)

    Sound wood Per g sound wood 231 34 409 168 17 5 4 3

    Decayed wood Per g sound wood 158 14 63 14 1 0.4 0.2 nd Per g decayed wood 522 47 207 47 3 1 1 nd

    ASL, acid-soluble lignin; nd, not detected.

    1092 D. J. Yelle, D. Wei, J. Ralph and K. E. Hammel

    Published 2011. This article is a US Government work and is in the public domain in the USA. Environmental Microbiology, 13, 1091–1100

  • Depletion of arylglycerol-b-aryl ether structures in the lignin

    Initially, we obtained two-dimensional one-bond 1H–13C heteronuclear single quantum coherence (HSQC) NMR spectra of the acetylated wood samples. This technique generates cross-peaks that correlate hydrogens in a mol- ecule with carbon

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