Investigation of the role played by cellobiose dehydrogenases from 1 Podospora anserina during lignocellulose degradation

Conversion of biomass into high-value products including biofuels is of great interest to develop sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated resulting in singly and multiple CDH mutants. We detected almost no difference in the growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, where a two-fold decrease in fertility of the mutants lacking PaCDH1 and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases vs . only one for the wild type. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. Addition of P. anserina LPMO to either wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass rely on different types of mechanisms in fungi. complex process involving dozens of enzymes. The roles of each enzyme or enzyme class not fully understood and utilization of a model amenable to genetic analysis the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose, yet seems to play a minor role on actual substrates such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensate lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO. CDH haem (24-26). haem cellulose role of CDH, we performed a thorough analysis of the role of the three enzymes encoded by the genome of the filamentous ascomycete P. anserina . This filamentous fungus is easy to manipulate and is able to rapidly complete its lifecycle with wood as sole carbon source (29-31). Gene deletions and construction of multiple mutants can rapidly be achieved. Moreover, genome sequencing (30) has revealed that its genome contains a large number of genes putatively involved in lignocellulose breakdown. Comparison with other fungi revealed that P. anserina has a high number of such genes like basidiomycetes (2, 30). Being genetically tractable and having a complement of genes implicated in lignocellulose breakdown similar to that of basidiomycetes make it thus a good model to decipher how fungi manage to breakdown lignocellulose. Here we investigate the in vivo role of three P. anserina genes encoding CDHs by targeted gene deletion and analyze phenotypes and secretomes of the mutants. thus to class IIB, PaCDH1 a CBM1 module its C-terminus thus to class IIA. These CDHs were previously characterized as able to oxidize cellobiose (12, 13) and to provide electrons to P. anserina AA9 LPMOs for efficient cleavage of cellulose (13). Intriguingly, PaCDH3 CBM1 cytochrome and PaCDH2 PaMpk1 PaMpk2 to a signaling pathway involved in numerous aspects of anserina lifecycle, of degradation and microarray analysis assays, hypothesized assayed DAB assay anserina anserina hyphal chrysogenum hyphae CDH mutants. CDHs a role in peroxide/superoxide P. anserina by DAB NBT assays. in the regulation of the expression of β -glucosidases in P. anserina Moreover, suggested that CDH could enhance cellulase activity by relieving product inhibition of cellobiohydrolases through the oxidation of cellobiose to cellobionic acid (54). P. anserina may compensate the lack of CDH with the secretion of β -glucosidases to favor GH6 and GH7 cellobiohydrolase activity by relieving product inhibition. This mechanism might in part explain the weak phenotypes of the CDH deletion mutants. these provide evidence that other redox partners than CDH are present in the CDH Δ mutant secretomes oxidative cleavage of cellulose by PaLPMO9H. One can suggest gallic or tannic acids as a potential candidate. Indeed, these acids have been shown to be alternative electron donors for LPMOs (50) and a the putative tannase/esterase that could be involved in the production of these molecules is degradation. Phylogenetic prediction of the function of this flavo-oxidase is hampered by the few characterized members of the AA3_2 subfamily (55). Our findings are in agreement with recent findings showing thatAA3_2 GMC oxidoreductases can serve as extracellular electron sources for LPMOs (48, 49) thus extending the array of fungal redox partners in filamentous fungi. Overall, the present data suggest that compensatory mechanisms indeed enable P. anserina to cope in most part with lack of CDH. Analysis by targeted gene deletion of the CDH genes of P. anserina has shown that these enzymes are important for degradation of crystalline cellulose, but that their absence does not result in a dramatic decrease in the ability to use complex biomasses such as wood shavings or miscanthus, in which crystalline cellulose may represent a minor fraction. The efficiency of mutant CDH Δ secretome to degrade cellulose appeared similar to that of wild-type, except that twice as much cellobiose was obtained as expected from the lack of conversion by CDH. Analysis of oxidized products indicated that LPMO are still active suggesting that additional partners able to provide electrons to LPMOs are present in CDH Δ . Based on its increased quantity in CDH Δ secretome, the AA3_2 flavo-oxidase Pa_5_5180/CAP65285 may be a good candidate for such a task. These data underscore the complex mechanisms of plant biomass degradation by fungi in which many activities, some potentially redundant, act in concert to achieve efficient degradation. This is particularly important for coprophilous fungi, such as P. anserina , because they likely encounter biomasses of diverse origins, which have in addition been more or less extensively digested by herbivores, but also the other competing fungi that grow and fructify in a succession on dung.