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

International audienceFilamentous 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 setup 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

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

International audienceBackground: 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 beta-(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

A fungal family of lytic polysaccharide monooxygenase-like copper proteins

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

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

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.Peer reviewe

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-ef-fective 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 oxida-tive 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

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

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 272μg 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 condition

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

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), PcAA14B, with the recently discovered glucuronoxylan-specific xylanase TtXyn30A, of the (sub)family GH30_7, displaying xylobiohydrolaseactivity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.Results: PcAA14B and TtXyn30A showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. PcAA14B was able to increase the release of xylobiose from TtXyn30A, 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 PcAA14B–TtXyn30A synergism was more prominent on substrates with low hemicellulose content, indicating that PcAA14B 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, PcAA14B 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 PcAA14B, and the GH30_7 glucuronoxylan-active xylobiohydrolase TtXyn30A, but also among PcAA14B and cellobiohydrolases. We hypothesize that PcAA14B creates free ends in the xylan polymer, which can be used as targets for the action of TtXyn30A. 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 biomas

Efficient biomass pretreatment using the White-rot Fungus Polyporus Brumalis.

Implementation of cheap and eco-friendly biomass pretreatment processes is necessary to develop sustainable biorefineries. In nature, white-rot basidiomycetes are able to degrade lignin efficiently and selectively and are thus of great interest in such bioprocesses. In this study, five basidiomycetes strains were evaluated for their ability to pretreat wheat straw under solid state fermentation. Fungal pretreatments were carried out in glass-column reactors under operating conditions approaching industrial practices. The pretreatment efficiency was evaluated through the quantification of dry weight losses and subsequent hydrolysis of the carbohydrate fraction by enzymatic cocktails. The highest lignin to cellulose losses ratio was obtained using a strain of Polyporus brumalis which exhibited high ligninolytic capabilities. This selectivity along with the low dry weight loss makes the pretreatment profitable by enhancing cellulose and hemicellulose conversion yields. Therefore P. brumalis can be viewed as a promising strain to pretreat lignocellulosic biomass for biorefinery applications

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

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 72h 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

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

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 H2O/g at 27°C for 15–16 days led to an improvement of carbohydrate accessibility with minimal matter loss