Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.
LPMOs are mono-copper enzymes that oxidatively degrade various polysaccharides. Genes encoding LPMOs in the AA9 family are abundant in filamentous fungi while their multiplicity remains elusive. We describe a detailed functional characterization of six AA9 LPMOs from the ascomycetous fungus Thermothielavioides terrestris LPH172 (syn. Thielavia terrestris ). These six LPMOs were shown to be upregulated during growth on different lignocellulosic substrates in our previous study. Here we produced them heterologously in Pichia pastoris and tested their activity on various model and native plant cell wall substrates. All six Tt AA9 LPMOs produced hydrogen peroxide in the absence of polysaccharide substrate and displayed peroxidase-like activity on a model substrate, yet only five of them were active on selected cellulosic substrates. Tt LPMO9A and Tt LPMO9E were also active on birch acetylated glucuronoxylan, but only when the xylan was combined with phosphoric acid-swollen cellulose (PASC). Another of the six AA9s, Tt LPMO9G, was active on spruce arabinoglucuronoxylan mixed with PASC. Tt LPMO9A, Tt LPMO9E, Tt LPMO9G and Tt LPMO9T could degrade tamarind xyloglucan and beechwood xylan when combined with PASC. Interestingly, none of the tested enzymes were active on wheat arabinoxylan, konjac glucomannan, acetylated spruce galactoglucomannan, or cellopentaose. Overall, these functional analyses support the hypothesis that the multiplicity of the fungal LPMO genes assessed in this study relates to the complex and recalcitrant structure of lignocellulosic biomass. Our study also highlights the importance of using native substrates in functional characterization of LPMOs as we were able to demonstrate distinct, previously unreported xylan-degrading activities of AA9 LPMOs using such substrates. Importance The discovery of LPMOs in 2010 has revolutionized the industrial biotechnology field, mainly by increasing the efficiency of cellulolytic enzyme cocktails. Nonetheless, the biological purpose for the multiplicity of LPMO-encoding genes in filamentous fungi has remained an open question. Here, we address this point by showing that six AA9 LPMOs from a single fungal strain have varying substrate preferences and activities on tested cellulosic and hemicellulosic substrates, including several native xylan substrates. Importantly, several of these activities could only be detected when using co-polymeric substrates that likely resemble plant cell walls, more than single fractionated polysaccharides do. Our results suggest that LPMOs have evolved to contribute to the degradation of different complex structures in plant cell walls where different biomass polymers are closely associated. This knowledge together with the elucidated novel xylanolytic activities could aid in further optimization of enzymatic cocktails for efficient degradation of lignocellulosic substrates and more.
Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose, and some also act on hemicelluloses, primarily other (substituted) β-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a handful AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Nc LPMO9F and the phylogenetically related, hitherto uncharacterized Nc LPMO9L from Neurospora crassa are active on both cellulose and cellulose-associated glucuronoxylan, but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that Nc LPMO9F preferentially cleaves xylan when acting on a cellulose–beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, Nc LPMO9L and previously characterized Mc LPMO9H from Malbranchea cinnamomea showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity towards glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path towards discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. Importance Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient co-polymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remains unclear. Here we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose–glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility, and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, co-polymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.
Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.
Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass will promote the use of this renewable material. Here, we have studied several process parameters (reaction temperature, type of enzyme blend, type of substrate, type of reductant, and in situ supply of hydrogen peroxide) to better understand how saccharification could be optimized, focusing on the role of lytic polysaccharide monooxygenases (LPMOs). Comparison of a simple, LPMO-rich cellulolytic secretome from the thermophilic fungus Thermoascus aurantiacus with the commercial cellulase preparation Cellic CTec2 showed that saccharification of (lignin-poor) sulfite-pulped spruce at 60 °C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 °C. Quantification of LPMO products showed that while LPMO activity contributed to saccharification efficiency, high levels of LPMO activity were not necessarily beneficial. Reactions with steam-exploded birch, rich in redox-active lignin, highlighted a strong impact of the feedstock on enzyme performance. In this case, the reaction with Cellic CTec2 at 50 °C was clearly most efficient. At 60 °C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H 2 O 2 -generating glucose oxidase to reactions with Cellic CTec2 at 50 °C led to strongly increased LPMO activity and, only for reactions with the lignin-poor substrate, improved saccharification yields. These results underpin the potential of the T. aurantiacus secretome for hydrolysis of lignin-poor substrates, and the usefulness of glucose oxidase for optimizing their saccharification. They also show that the efficiency of LPMO-containing cellulase preparations is highly dependent on the nature of the reductant and the substrate.
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