Structure and function of lytic polysaccharide monooxygenases (LPMOS)and other redox enzymes involved in biomass processing

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has revolutionized our understanding of biomass conversion in Nature and has been instrumental for the development of economically sustainable lignocellulose biorefineries. LPMOs are mono-copper redox enzymes that attack the most recalcitrant parts of biopolymers such as crystalline cellulose and chitin. LPMOs employ the power of redox chemistry to cleave glycosidic bonds that are not easily cleaved by hydrolytic enzymes. By doing so, they make the substrate more tractable to the action of canonical enzymes such as endo- and exo-cellulases. LPMOs are abundant in Nature, for example in the secretomes of wood-decaying fungi. Despite their importance in both Nature and the biorefinery, several aspects of these intriguing enzymes remain unclear. The catalytic mechanism of LPMOs is of particular importance because the enzymes display a unique active site architecture that is employed to catalyze a challenging chemical reaction on a substrate that is embedded in a crystalline lattice. Deeper insight into this mechanism may have wide-reaching consequences, not only for biomass processing but also, perhaps, in developing enzymatic or other catalytic systems for difficult reactions, such as controlled oxidation of methane and other alkanes. Using a variety of experimental approaches, we are studying LPMO function, addressing issues such as the structural basis of oxidative regio-selectivity and substrate specificity, routes and mechanisms for electron delivery, the roles of appended carbohydrate-binding domains, and the determinants of catalytic activity and stability. Knowledge gained from these fundamental studies is being used to optimize biomass conversion processes, whereas translation of this knowledge to other fields is also being explored. In this presentation, I will review recent work in the field and present our latest results. Special attention will be paid to recent research in our group that has led to the proposal that LPMOs may not be true monooxygenases. Based on our recent results, we have proposed that hydrogen peroxide, rather than molecular oxygen, is the preferred co-substrate of LPMOs. While this paradigm-shattering proposal may not yet have found wide acceptance in the field, we have already demonstrated that the controlled administration of hydrogen peroxide during biomass degradation by LPMO-containing commercial cellulolytic enzyme cocktails leads to drastically improved LPMO activity and more efficient saccharification. These recent findings also shed new light on the interplay between LPMOs and other redox enzymes in the secretomes of biomass degrading microorganisms

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

Recovering Homography from Camera Captured Documents using Convolutional Neural Networks

Removing perspective distortion from hand held camera captured document images is one of the primitive tasks in document analysis, but unfortunately, no such method exists that can reliably remove the perspective distortion from document images automatically. In this paper, we propose a convolutional neural network based method for recovering homography from hand-held camera captured documents. Our proposed method works independent of document's underlying content and is trained end-to-end in a fully automatic way. Specifically, this paper makes following three contributions: Firstly, we introduce a large scale synthetic dataset for recovering homography from documents images captured under different geometric and photometric transformations; secondly, we show that a generic convolutional neural network based architecture can be successfully used for regressing the corners positions of documents captured under wild settings; thirdly, we show that L1 loss can be reliably used for corners regression. Our proposed method gives state-of-the-art performance on the tested datasets, and has potential to become an integral part of document analysis pipeline.Comment: 10 pages, 8 figure

Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1

<p>Abstract</p> <p>Background</p> <p><it>Lactobacillus plantarum </it>is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for <it>in situ </it>delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.</p> <p>Results</p> <p>We have constructed a library of 76 Sec-type signal peptides from <it>L. plantarum </it>WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA.</p> <p>Conclusion</p> <p>This is the first comprehensive experimental study showing that predicted SPs in the <it>L. plantarum </it>genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in <it>L. plantarum</it>. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.</p

Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara

Background Cellulases play a key role in the enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. Thus, the discovery of novel cellulases from exotic biological niches is of great interest as they may present properties that are valuable in the biorefning of lignocellulosic biomass. Results We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multi-domain enzyme from the unusual gill endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The catalytic GH5 domain, was cloned and recombinantly produced with or without a C-terminal family 10 carbohydrate-binding module (CBM). Both variants showed hydrolytic endo-activity on soluble substrates such as β-glucan, carboxymethylcellulose and konjac glucomannan, respectively. However, low activity was observed towards the crystalline form of cellulose. Interestingly, when co-incubated with a cellulose-active LPMO, a clear syn‑ ergy was observed that boosted the overall hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 Å resolution and revealed a substrate binding cleft extension containing a putative+3 subsite, which is uncommon in this enzyme family. The enzyme was active in a wide range of pH, temperatures and showed high tolerance for NaCl. Conclusions This study provides significant knowledge in the discovery of new enzymes from shipworm gill endo‑ symbionts and sheds new light on biochemical and structural characterization of cellulolytic cellulase. Study demon‑ strated a boost in the hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose-active LPMO. These findings will be relevant for the development of future enzyme cocktails that may be useful for the biotechnological conversion of lignocellulose

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 -&gt; 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

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.info:eu-repo/semantics/publishe

Identification of proteins related to the stress response in Enterococcus faecalis V583 caused by bovine bile

<p>Abstract</p> <p>Background</p> <p><it>Enterococcus faecalis </it>is an opportunistic pathogen and one of the most important causes of hospital infections. Bile acids are a major stress factor bacteria have to cope with in order to colonize and survive in the gastro-intestinal tract. The aim of this study was to investigate the effects of bile acids on the intracellular proteome of <it>E. faecalis </it>V583.</p> <p>Results</p> <p>The proteomes of cells challenged with 1% bile were analyzed after 20 - 120 minutes exposure, using 2D gel electrophoresis and mass spectrometry. Among the approximately 500 observed proteins, 53 unique proteins were found to be regulated in response to bile and were identified with mass spectrometry. The identified proteins belonged to nine different functional classes, including fatty acid- and phospholipid-biosynthesis, energy metabolism, and transport and binding. Proteins involved in fatty acid and phospholipid biosynthesis pathways were clearly overrepresented among the identified proteins and all were down-regulated upon exposure to bile. The proteome data correlated reasonably well with data from previous transcriptome experiments done under the same conditions, but several differences were observed.</p> <p>Conclusion</p> <p>The results provide an overview of potentially important proteins that <it>E. faecalis </it>V583 needs to regulate in order to survive and adapt to a bile-rich environment, among which are several proteins involved in fatty acid and phospholipid biosynthesis pathways. In addition, this study reveals several hypothetical proteins, which are both abundant and clearly regulated and thus stand out as targets for future studies on bile stress.</p