Publications by authors named "Sacha Grisel"

34 Publications

Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs).

J Biol Chem 2020 Nov 24;296:100086. Epub 2020 Nov 24.

INRAE, Aix-Marseille University, Polytech Marseille, UMR1163 BBF, Marseille, France. Electronic address:

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides, thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose, but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides, and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharide degradation suggest that the subtle differences in amino-acid sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO subclusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.015545DOI Listing
November 2020

Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs).

J Biol Chem 2020 Nov 16. Epub 2020 Nov 16.

BBF, INRAE, France.

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.015545DOI Listing
November 2020

AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis cinerea.

Mol Plant Pathol 2020 12 7;21(12):1620-1633. Epub 2020 Oct 7.

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Rome, Italy.

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mpp.13002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694680PMC
December 2020

Evaluation of the Enzymatic Arsenal Secreted by During Growth on Sugarcane Bagasse With a Focus on LPMOs.

Front Bioeng Biotechnol 2020 25;8:1028. Epub 2020 Aug 25.

INRAE, Faculté des Sciences de Luminy, Aix Marseille Université, UMR 1163 Biodiversité et Biotechnologie Fongiques, Polytech Marseille, Marseille, France.

The high demand for energy and the increase of the greenhouse effect propel the necessity to develop new technologies to efficiently deconstruct the lignocellulosic materials into sugars monomers. Sugarcane bagasse is a rich polysaccharide residue from sugar and alcohol industries. The thermophilic fungus (syn. ) is an interesting model to study the enzymatic degradation of biomass. The genome of encodes an extensive repertoire of cellulolytic enzymes including 23 lytic polysaccharide monooxygenases (LPMOs) from the Auxiliary Activity family 9 (AA9), which are known to oxidatively cleave the β-1,4 bonds and boost the cellulose conversion in a biorefinery context. To achieve a deeper understanding of the enzymatic capabilities of on sugarcane bagasse, we pretreated this lignocellulosic residue with different methods leading to solids with various cellulose/hemicellulose/lignin proportions and grew on these substrates. The secreted proteins were analyzed using proteomics taking advantage of two mass spectrometry methodologies. This approach unraveled the secretion of many CAZymes belonging to the Glycosyl Hydrolase (GH) and AA classes including several LPMOs that may contribute to the biomass degradation observed during fungal growth. Two AA9 LPMOs, called LPMO9B and LPMO9H, were selected from secretomic data and enzymatically characterized. Although LPMO9B and LPMO9H were both active on cellulose, they differed in terms of optimum temperatures and regioselectivity releasing either C1 or C1-C4 oxidized oligosaccharides, respectively. LPMO activities were also measured on sugarcane bagasse substrates with different levels of complexity. The boosting effect of these LPMOs on bagasse sugarcane saccharification by a commercial cocktail was also observed. The partially delignified bagasse was the best substrate considering the oxidized oligosaccharides released and the acid treated bagasse was the best one in terms of saccharification boost.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.01028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477043PMC
August 2020

A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan.

Biotechnol Biofuels 2020 10;13:142. Epub 2020 Aug 10.

Industrial Biotechnology and Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.

Background: Hemicellulose accounts for a significant part of plant biomass, and still poses a barrier to the efficient saccharification of lignocellulose. The recalcitrant part of hemicellulose is a serious impediment to the action of cellulases, despite the use of xylanases in the cellulolytic cocktail mixtures. However, the complexity and variety of hemicelluloses in different plant materials require the use of highly specific enzymes for a complete breakdown. Over the last few years, new fungal enzymes with novel activities on hemicelluloses have emerged. In the present study, we explored the synergistic relationships of the xylan-active AA14 lytic polysaccharide monooxygenase (LPMO), AA14B, with the recently discovered glucuronoxylan-specific xylanase Xyn30A, of the (sub)family GH30_7, displaying xylobiohydrolase activity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.

Results: AA14B and Xyn30A showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. AA14B was able to increase the release of xylobiose from Xyn30A, showing a degree of synergism (DS) of 3.8 on birchwood cellulosic fibers, and up to 5.7 on pretreated beechwood substrates. The increase in activity was dose- and time- dependent. A screening study on beechwood materials pretreated with different methods showed that the effect of the AA14B-Xyn30A synergism was more prominent on substrates with low hemicellulose content, indicating that AA14B is mainly active on the recalcitrant part of xylan, which is in close proximity to the underlying cellulose fibers. Simultaneous addition of both enzymes resulted in higher DS than sequential addition. Moreover, AA14B was found to enhance cellobiose release from cellobiohydrolases during hydrolysis of pretreated lignocellulosic substrates, as well as microcrystalline cellulose.

Conclusions: The results of the present study revealed a new synergistic relationship not only among two recently discovered xylan-active enzymes, the LPMO AA14B, and the GH30_7 glucuronoxylan-active xylobiohydrolase Xyn30A, but also among AA14B and cellobiohydrolases. We hypothesize that AA14B creates free ends in the xylan polymer, which can be used as targets for the action of Xyn30A. The results are of special importance for the design of next-generation enzymatic cocktails, able to efficiently remove hemicelluloses, allowing complete saccharification of cellulose in plant biomass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-020-01777-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419196PMC
August 2020

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

DNA Res 2020 Apr;27(2)

INRAE, UMR1163, Biodiversity and Biotechnology of Fungi, Aix Marseille University, 13009 Marseille, France.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/dnares/dsaa011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406137PMC
April 2020

A fungal family of lytic polysaccharide monooxygenase-like copper proteins.

Nat Chem Biol 2020 03 13;16(3):345-350. Epub 2020 Jan 13.

INRA, Biodiversité et Biotechnologie Fongiques (BBF), UMR1163, Aix Marseille Université, Marseille, France.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-019-0438-8DOI Listing
March 2020

Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates.

Biotechnol Biofuels 2019 3;12:206. Epub 2019 Sep 3.

2Biodiversité et Biotechnologie Fongiques, UMR1163, INRA, Aix Marseille Université, Marseille, France.

Background: Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood.

Results: In this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from (LPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of LPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of LPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network.

Conclusions: Absence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and non-oxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-019-1548-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721207PMC
September 2019

Lytic polysaccharide monooxygenases (LPMOs) facilitate cellulose nanofibrils production.

Biotechnol Biofuels 2019 24;12:156. Epub 2019 Jun 24.

1UR1268 Biopolymères Interactions Assemblages, INRA, 44316 Nantes, France.

Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC).

Results: The fungal LPMO from AA9 family (LPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with LPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC.

Conclusions: This study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-019-1501-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589874PMC
June 2019

AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes.

Biotechnol Biofuels 2019 16;12:55. Epub 2019 Mar 16.

1Biodiversité et Biotechnologie Fongiques, UMR1163, INRA, Aix Marseille Université, Marseille, France.

Background: Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete , but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity.

Results: In this study, the enzymatic cocktails secreted by five strains from the genus ( strains BRFM 405, 1487, 1489, 1490 and strain BRFM 430) were tested for their ability to boost a reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from (AaAA16) was produced to high level in LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of CBHI on cellulosic substrates.

Conclusions: Although spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-019-1394-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420742PMC
March 2019

Integrative visual omics of the white-rot fungus exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass.

Biotechnol Biofuels 2018 23;11:201. Epub 2018 Jul 23.

1Aix Marseille Univ, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Background: Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide.

Results: We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners.

Conclusion: As a peculiar feature of , we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-018-1198-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055342PMC
July 2018

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Nat Chem Biol 2018 03 29;14(3):306-310. Epub 2018 Jan 29.

INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2558DOI Listing
March 2018

Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type.

Sci Rep 2017 12 19;7(1):17792. Epub 2017 Dec 19.

FARE Laboratory, INRA, University of Reims Champagne-Ardenne, 51100, Reims, France.

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-17938-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736606PMC
December 2017

Biological wheat straw valorization: Multicriteria optimization of Polyporus brumalis pretreatment in packed bed bioreactor.

Microbiologyopen 2018 02 27;7(1). Epub 2017 Oct 27.

Aix-Marseille Univ, INRA, BBF, Biodiversité et Biotechnologie Fongiques, Marseille, France.

The purpose of this work was to optimize the pretreatment process of wheat straw by Polyporus brumalis_BRFM985 in order to improve carbohydrate accessibility for more efficient bioconversion. Indeed, there is growing demands to develop sustainable routes for lignocellulosic feedstocks valorization into value-added products in energy, chemicals, materials, and animal feed fields. To be achieved, implementation of cheap and ecofriendly biomass pretreatment processes is necessary. In this frame, white rot basidiomycetes, well known for their ability to degrade lignin efficiently and selectively, are of great interest. The pretreatment of wheat straw by Polyporus brumalis_BRFM985 was performed in packed bed bioreactor and optimized using response surface methodology. The four pretreatment parameters optimized were metals addition (Cu, Mn, and Fe), time of culture, initial water content, and temperature. Multicriteria optimization highlighted that wheat straw pretreatment by Polyporus brumalis_BRFM985 in the presence of metals with high initial water content of 3.6 g H O/g at 27°C for 15-16 days led to an improvement of carbohydrate accessibility with minimal matter loss.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mbo3.530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5822346PMC
February 2018

The yeast encodes functional lytic polysaccharide monooxygenases.

Biotechnol Biofuels 2017 12;10:215. Epub 2017 Sep 12.

INRA, Aix Marseille University BBF, Biodiversité et Biotechnologie Fongiques, 13288 Marseille, France.

Background: Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that have revolutionized our understanding of lignocellulose degradation. Fungal LPMOs of the AA9 family target cellulose and hemicelluloses. AA9 LPMO-coding genes have been identified across a wide range of fungal saprotrophs (Ascomycotina, Basidiomycotina, etc.), but so far they have not been found in more basal lineages. Recent genome analysis of the yeast (Saccharomycotina) revealed the presence of several LPMO genes, which belong to the AA9 family.

Results: In this study, three AA9 LPMOs from were successfully produced and biochemically characterized. The use of native signal peptides was well suited to ensure correct processing and high recombinant production of LPMO9A, LPMO9B, and LPMO9C in . We show that LPMO9A and LPMO9B were both active on cellulose and xyloglucan, releasing a mixture of soluble C1- and C4-oxidized oligosaccharides from cellulose. All three enzymes disrupted cellulose fibers and significantly improved the saccharification of pretreated lignocellulosic biomass upon addition to a commercial cellulase cocktail.

Conclusions: The unique enzymatic arsenal of compared to other yeasts could be beneficial for plant cell wall decomposition in a saprophytic or pathogenic context. From a biotechnological point of view, LPMOs are promising candidates to further enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-017-0903-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596469PMC
September 2017

The integrative omics of white-rot fungus Pycnoporus coccineus reveals co-regulated CAZymes for orchestrated lignocellulose breakdown.

PLoS One 2017 10;12(4):e0175528. Epub 2017 Apr 10.

Aix-Marseille Université, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Innovative green technologies are of importance for converting plant wastes into renewable sources for materials, chemicals and energy. However, recycling agricultural and forestry wastes is a challenge. A solution may be found in the forest. Saprotrophic white-rot fungi are able to convert dead plants into consumable carbon sources. Specialized fungal enzymes can be utilized for breaking down hard plant biopolymers. Thus, understanding the enzymatic machineries of such fungi gives us hints for the efficient decomposition of plant materials. Using the saprotrophic white-rot fungus Pycnoporus coccineus as a fungal model, we examined the dynamics of transcriptomic and secretomic responses to different types of lignocellulosic substrates at two time points. Our integrative omics pipeline (SHIN+GO) enabled us to compress layers of biological information into simple heatmaps, allowing for visual inspection of the data. We identified co-regulated genes with corresponding co-secreted enzymes, and the biological roles were extrapolated with the enriched Carbohydrate-Active Enzyme (CAZymes) and functional annotations. We observed the fungal early responses for the degradation of lignocellulosic substrates including; 1) simultaneous expression of CAZy genes and secretion of the enzymes acting on diverse glycosidic bonds in cellulose, hemicelluloses and their side chains or lignin (i.e. hydrolases, esterases and oxido-reductases); 2) the key role of lytic polysaccharide monooxygenases (LPMO); 3) the early transcriptional regulation of lignin active peroxidases; 4) the induction of detoxification processes dealing with biomass-derived compounds; and 5) the frequent attachments of the carbohydrate binding module 1 (CBM1) to enzymes from the lignocellulose-responsive genes. Our omics combining methods and related biological findings may contribute to the knowledge of fungal systems biology and facilitate the optimization of fungal enzyme cocktails for various industrial applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386290PMC
September 2017

Visual Comparative Omics of Fungi for Plant Biomass Deconstruction.

Front Microbiol 2016 24;7:1335. Epub 2016 Aug 24.

Aix-Marseille Université, INRA, UMR1163, Biodiversité et Biotechnologie Fongiques Marseille, France.

Wood-decay fungi contain the cellular mechanisms to decompose such plant cell wall components as cellulose, hemicellulose, and lignin. A multi-omics approach to the comparative analysis of wood-decay fungi gives not only new insights into their strategies for decomposing recalcitrant plant biomass, but also an understanding of how to exploit these mechanisms for biotechnological applications. We have developed an analytical workflow, Applied Biomass Conversion Design for Efficient Fungal Green Technology (ABCDEFGT), to simplify the analysis and interpretation of transcriptomic and secretomic data. ABCDEFGT utilizes self-organizing maps for grouping genes with similar transcription patterns, and an overlay with secreted proteins. The key feature of ABCDEFGT is simple graphic outputs of genome-wide transcriptomic and secretomic topographies, which enables visual inspection without a priori of the omics data and facilitates discoveries of co-regulated genes and proteins. Genome-wide omics landscapes were built with the newly sequenced fungal species Pycnoporus coccineus, Pycnoporus sanguineus, and Pycnoporus cinnabarinus grown on various carbon sources. Integration of the post-genomic data revealed a global overlap, confirming the pertinence of the genome-wide approach. ABCDEFGT was evaluated by comparison with the latest clustering method for ease of output interpretation, and ABCDEFGT gave a better biological representation of fungal behaviors. The genome-wide multi-omics strategy allowed us to determine the potential synergy of particular enzymes decomposing cellulose, hemicellulose, and lignin such as Lytic Polysaccharide Monooxygenases, modular enzymes associated with a cellulose binding module1, and Class II Peroxidase isoforms co-regulated with oxido-reductases. Overall, ABCDEFGT was capable of visualizing genome-wide transcriptional and secretomic profiles for intuitive interpretations and is suitable for exploration of newly-sequenced organisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2016.01335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996036PMC
September 2016

Characterization of a new aryl-alcohol oxidase secreted by the phytopathogenic fungus Ustilago maydis.

Appl Microbiol Biotechnol 2016 Jan 9;100(2):697-706. Epub 2015 Oct 9.

INRA, UMR1163 BBF, Polytech'Marseille, F-13288, Marseille, France.

The discovery of novel fungal lignocellulolytic enzymes is essential to improve the breakdown of plant biomass for the production of second-generation biofuels or biobased materials in green biorefineries. We previously reported that Ustilago maydis grown on maize secreted a diverse set of lignocellulose-acting enzymes including hemicellulases and putative oxidoreductases. One of the most abundant proteins of the secretome was a putative glucose-methanol-choline (GMC) oxidoreductase. The phylogenetic prediction of its function was hampered by the few characterized members within its clade. Therefore, we cloned the gene and produced the recombinant protein to high yield in Pichia pastoris. Functional screening using a library of substrates revealed that this enzyme was able to oxidize several aromatic alcohols. Of the tested aryl-alcohols, the highest oxidation rate was obtained with 4-anisyl alcohol. Oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol can serve as electron acceptors. This GMC oxidoreductase displays the characteristics of an aryl-alcohol oxidase (E.C.1.1.3.7), which is suggested to act on the lignin fraction in biomass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-015-7021-3DOI Listing
January 2016

Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Front Microbiol 2015 23;6:1002. Epub 2015 Sep 23.

Architecture et Fonction des Macromolècules Biologiques, CNRS-Aix-Marseille University UMR 7257 Marseille, France ; INRA, USC 1408 AFMB Marseille, France.

Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2015.01002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585289PMC
October 2015

Solid-state fermentation in multi-well plates to assess pretreatment efficiency of rot fungi on lignocellulose biomass.

Microb Biotechnol 2015 Nov 6;8(6):940-9. Epub 2015 Aug 6.

INRA, UMR 1163 Biodiversity and Biotechnology of Fungi, F-13009, Marseille, France.

The potential of fungal pretreatment to improve fermentable sugar yields from wheat straw or Miscanthus was investigated. We assessed 63 fungal strains including 53 white-rot and 10 brown-rot fungi belonging to the Basidiomycota phylum in an original 12 day small-scale solid-state fermentation (SSF) experiment using 24-well plates. This method offers the convenience of one-pot processing of samples from SSF to enzymatic hydrolysis. The comparison of the lignocellulolytic activity profiles of white-rot fungi and brown-rot fungi showed different behaviours. The hierarchical clustering according to glucose and reducing sugars released from each biomass after 72 h enzymatic hydrolysis splits the set of fungal strains into three groups: efficient, no-effect and detrimental-effect species. The efficient group contained 17 species belonging to seven white-rot genera and one brown-rot genus. The yield of sugar released increased significantly (max. 62%) compared with non-inoculated controls for both substrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1751-7915.12307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621447PMC
November 2015

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Biotechnol Biofuels 2015 20;8:90. Epub 2015 Jun 20.

INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, ESIL Polytech, F-13288 Marseille, France.

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-015-0274-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487207PMC
July 2015

A PCR-based method to quantify fungal growth during pretreatment of lignocellulosic biomass.

J Microbiol Methods 2015 Aug 30;115:67-70. Epub 2015 May 30.

INRA, UMR1163 Biodiversity and Biotechnology of Fungi, F-13009 Marseille, France; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversity and Biotechnology of Fungi, F-13009 Marseille, France. Electronic address:

Filamentous fungi have shown great potential in the pretreatment of lignocellulosic biomass and their use in bio-processes based on Solid State Fermentation (SSF) opens promising perspectives for plant biomass valorization. Obviously, quantification of the fungal biomass throughout the fermentation is a crucial parameter for SSF evaluation and control, both at the laboratory and industrial scale. Here we provide a qPCR-based method as a reliable alternative to conventional methods to estimate mycelial growth during plant biomass treatment. For the three strains analyzed, the lowest detection limit ranged from 58 to 272 μg mycelium dry weight per gram biomass (dry weight). We show that the qPCR-based method allows fungal growth monitoring during fermentation and provides relevant information for selection of the most appropriate fungal strains in specific SSF/reactor conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mimet.2015.05.024DOI Listing
August 2015

Laccase/HBT and laccase-CBM/HBT treatment of softwood kraft pulp: impact on pulp bleachability and physical properties.

Bioresour Technol 2012 Oct 4;121:68-75. Epub 2012 Jul 4.

INRA, UMR 1163 BCF, 13288 Marseille, France.

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biortech.2012.06.077DOI Listing
October 2012

Exploring the natural fungal biodiversity of tropical and temperate forests toward improvement of biomass conversion.

Appl Environ Microbiol 2012 Sep 6;78(18):6483-90. Epub 2012 Jul 6.

INRA, UMR1163 BCF, Marseille, France.

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.01651-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426683PMC
September 2012

Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw.

Bioresour Technol 2012 Jun 10;114:589-96. Epub 2012 Mar 10.

INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France.

Fusarium verticillioides secretes enzymes (secretome), some of which might be potentially useful for saccharification of lignocellulosic biomass since supplementation of commercial cellulases from Trichoderma reesei with the F. verticillioides secretome improved the enzymatic release of glucose, xylose and arabinose from wheat straw by 24%, 88% and 68%, respectively. Determination of enzymatic activities revealed a broad range of hemicellulases and pectinases poorly represented in commercial cocktails. Proteomics approaches identified 57 proteins potentially involved in lignocellulose breakdown among a total of 166 secreted proteins. This analysis highlighted the presence of carbohydrate-active enzymes (CAZymes) targeting pectin (from glycoside hydrolase families GH5, GH27, GH28, GH43, GH51, GH54, GH62, GH88 and GH93, polysaccharide lyase family PL4 and carbohydrate esterase family CE8) and hemicelluloses (from glycoside hydrolase families GH3, GH10, GH11, GH30, GH39, GH43 and GH67). These data provide a first step towards the identification of candidates to supplement T. reesei enzyme preparations for lignocellulose hydrolysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biortech.2012.03.009DOI Listing
June 2012

Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching.

J Biotechnol 2009 Jul 4;142(3-4):220-6. Epub 2009 May 4.

INRA, UMR1163, Biotechnologie des Champignons Filamenteux, Case 932, 163 avenue de Luminy, F-13000 Marseille, France.

Pycnoporus cinnabarinus laccase was fused to the C-terminal linker and carbohydrate binding module (CBM) of Aspergillus niger cellobiohydrolase B (CBHB). The chimeric enzyme of molecular mass 100 kDa was successfully produced in A. niger. Laccase-CBM was further purified to determine its main biochemical properties. The Michaelis-Menten constant and pH activity profile were not modified, but the chimeric enzyme was less thermostable than either the P. cinnabarinus laccase or the recombinant laccase produced in the same strain. Laccase-CBM was able to bind to a cellulosic substrate and, to a greater extent, to softwood kraft pulp. Binding to the pulp was shown to be mainly time and temperature-dependent. Laccase-CBM was further investigated for its softwood kraft pulp biobleaching potential and compared with the P. cinnabarinus laccase. Addition of a CBM was shown to greatly improve the delignification capabilities of the laccase in the presence of 1-hydroxybenzotriazole (HBT). In addition, ClO(2) reduction using 5 U of chimeric enzyme per gram of pulp was almost double than that observed using 20 U of P. cinnabarinus laccase per gram of pulp. We demonstrated that conferring a carbohydrate binding capability to the laccase could significantly enhance its biobleaching properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiotec.2009.04.013DOI Listing
July 2009

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood.

Appl Microbiol Biotechnol 2008 Sep 25;80(4):719-33. Epub 2008 Jul 25.

INRA, UMR1163, Biotechnologie des Champignons Filamenteux, 13000, Marseille, France.

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-008-1596-xDOI Listing
September 2008

Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 Jun 12;63(Pt 6):495-8. Epub 2007 May 12.

Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d'Ingénieurs de Luminy-Case 925, Marseille CEDEX 9, France.

The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 A resolution and that were suitable for structure determination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1744309107020416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335076PMC
June 2007

Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Apr 25;62(Pt 4):409-11. Epub 2006 Mar 25.

Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d'Ingénieurs de Luminy-Case 925, 13288 Marseille CEDEX 9, France.

The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4(1)32 or P4(3)32, with unit-cell parameters a = b = c = 166.8 angstroms. Diffraction data were collected to a maximum resolution of 2.7 angstroms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1744309106009407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2222560PMC
April 2006

Expression, purification and crystallization of the SARS-CoV macro domain.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Apr 25;62(Pt 4):405-8. Epub 2006 Mar 25.

Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, UMR 6098-Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9, France.

Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1''-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182-355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 angstroms, beta = 91.4 degrees, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 angstroms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1744309106009274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2222557PMC
April 2006