The pyrroloquinoline-quinone (PQQ)-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea (CcPDH), belonging to the AA12 family, drives lytic polysaccharide monooxygenase (LPMO) action

Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs accelerate the decomposition of cellulose by cellulases by catalyzing the oxidative cleavage of glycosidic bonds after activation by an external electron donor (1-3). LPMOs procure electrons from non-enzymatic electron donors, such as ascorbic acid, lignin and other plant biomass-derived phenols (1-3), or they can be activated by flavin-dependent oxidoreductases, directly or through plant-derived diphenols and quinones acting as redox mediators (3-7). Cellobiose dehydrogenase, in particular, efficiently transfers electrons from its AA3_1 dehydrogenase domain to LPMOs via an appended AA8 cytochrome domain (7). Here we show that LPMOs can be activated by a quinohemoprotein, namely the pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase CcPDH from Coprinopsis cinerea, the founding member of the recently discovered AA12 family (8). CcPDH has a domain composition similar to that of cellobiose dehydrogenases (CDHs) but contains a central catalytic AA12 dehydrogenase domain, rather than an AA3_1 domain. We have studied the ability of full length CcPDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs, NcLPMO9F and NcLPMO9C. Our study shows that both the AA8 and CBM1 domains of CcPDH have a positive effect on the CcPDH-NcLPMO system. The interplay between the PDH and LPMOs seemed also to depend on whether the LPMO contained a CBM. Unlike the single dehydrogenase domain of MtCDH from Myriococcum thermophylum, the AA12 dehydrogenase domain of CcPDH could drive the LPMO reaction, which is due to the non-covalently bound PQQ co-factor acting as a diphenol/quinone redox mediator. CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool in future studies of LPMOs and redox enzyme systems involved in cellulose degradation. References: [1] Vaaje-Kolstad, G. et al. (2010) Science 330, 219-222. [2] Hemsworth, G. R., et al. (2015) Trends Biotechnol 33:747-761. [3] Kracher, D. et al. (2016) Science 352, 1098-1101. [4] Westereng, B. et al. (2015) Sci Rep 5:18561. [5] Langston, J.A. et al. (2011) Appl Environ Microbiol 77:7007-7015. [6] Garajova, S. et al. (2016) Sci Rep 6:28276. [7] Tan, T.C. et al. (2015) Nat Commun 6:7542. [8] Takeda, K. et al. (2015) PLoS One 10:e0115722

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

BACKGROUND: Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. RESULTS: In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3–5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. CONCLUSIONS: We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions

Carbohydrate-binding modules (CBMs) revisited. Reduced amount of water counterbalances the need for CBMs

Peer reviewe

Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa

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) beta-(1 -> 4)-glucans. Oxidative cleavage of xylan has been shown for only a few AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Neurospora crassa LPMO9F (NcLPMO9F) and the phylogenetically related, hitherto uncharacterized NcLPMO9L from N. 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 NcLPMO9F preferentially cleaves xylan when acting on a cellulosebeechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, NcLPMO9L and the previously characterized McLPMO9H, 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 xylanactive 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 toward glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path toward discovery of additional xylanactive 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 copolymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remain 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, copolymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs

Cellulases without carbohydrate-binding modules in high consistency ethanol production process

Peer reviewe

Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression

Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a “green enzyme,” laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda’s gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7–8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.publishedVersio

Effects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions

Background: Sugar cane internodes can be divided diagonally into four fractions, of which the two innermost ones are the least recalcitrant pith and the moderately accessible pith-rind interface. These fractions differ in enzymatic hydrolyzability due to structural differences. In general, cellulose hydrolysis in plants is hindered by its physical interaction with hemicellulose and lignin. Lignin is believed to be linked covalently to hemicellulose through hydroxycinnamic acids, forming a compact matrix around the polysaccharides. Acetyl xylan esterase and three feruloyl esterases were evaluated for their potential to fragment the lignocellulosic network in sugar cane and to indirectly increase the accessibility of cellulose. Results: The hydrolyzability of the pith and pith-rind interface fractions of a low-lignin-containing sugar cane clone (H58) was compared to that of a reference cultivar (RC). Acetyl xylan esterase enhanced the rate and overall yield of cellulose and xylan hydrolysis in all four substrates. Of the three feruloyl esterases tested, only TsFaeC was capable of releasing p-coumaric acid, while AnFaeA and NcFaeD released ferulic acid from both the pith and interface fractions. Ferulic acid release was higher from the less recalcitrant clone (H58)/fraction (pith), whereas more p-coumaric acid was released from the clone (RC)/fraction (interface) with a higher lignin content. In addition, a compositional analysis of the four fractions revealed that p-coumaroyl content correlated with lignin, while feruloyl content correlated with arabinose content, suggesting different esterification patterns of these two hydroxycinnamic acids. Despite the extensive release of phenolic acids, feruloyl esterases only moderately promoted enzyme access to cellulose or xylan. Conclusions: Acetyl xylan esterase TrAXE was more efficient in enhancing the overall saccharification of sugar cane, compared to the feruloyl esterases AnFaeA, TsFaeC, and NcFaeD. The hydroxycinnamic acid composition of sugar cane fractions and the hydrolysis data together suggest that feruloyl groups are more likely to decorate xylan, while p-coumaroyl groups are rather linked to lignin. The three different feruloyl esterases had distinct product profiles on non-pretreated sugar cane substrate, indicating that sugar cane pith could function as a possible natural substrate for feruloyl esterase activity measurements. Hydrolysis data suggest that TsFaeC was able to release p-coumaroyl groups esterifying lignin.Peer reviewe

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

publishedVersio