Inactivation of Cellobiose Dehydrogenases Modifies the ... · Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina Narumon

Inactivation of Cellobiose DehydrogenasesModifies the Cellulose DegradationMechanism of Podospora anserina

Narumon Tangthirasunun,a David Navarro,b Sona Garajova,b Didier Chevret,c

Laetitia Chan Ho Tong,a Valérie Gautier,a Kevin D. Hyde,d Philippe Silar,a

Jean-Guy Berrinb,c

University of Paris Diderot, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain, Paris,Francea; INRA, Aix Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques,Marseille, Franceb; INRA, UMR1319 Micalis, Plateforme d'Analyse Protéomique de Paris Sud-Ouest, Jouy-en-Josas, Francec; Center of Excellence in Fungal Research and School of Science, Mae Fah Luang University,Chiang Rai, Thailandd

ABSTRACT Conversion of biomass into high-value products, including biofuels, is ofgreat interest to developing sustainable biorefineries. Fungi are an inexhaustiblesource of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs)play an important role in the breakdown through synergistic action with fungal lyticpolysaccharide monooxygenases (LPMOs). The three CDH genes of the model fun-gus Podospora anserina were inactivated, resulting in single and multiple CDH mu-tants. We detected almost no difference in growth and fertility of the mutants onvarious lignocellulose sources, except on crystalline cellulose, on which a 2-fold de-crease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2was observed. A striking difference between wild-type and mutant secretomeswas observed. The secretome of the mutant lacking all CDHs contained fivebeta-glucosidases, whereas the wild type had only one. P. anserina seems tocompensate for the lack of CDH with secretion of beta-glucosidases. The addi-tion of P. anserina LPMO to either the wild-type or mutant secretome resulted inimprovement of cellulose degradation in both cases, suggesting that other redoxpartners present in the mutant secretome provided electrons to LPMOs. Overall,the data showed that oxidative degradation of cellulosic biomass relies on differ-ent types of mechanisms in fungi.

IMPORTANCE Plant biomass degradation by fungi is a complex process involvingdozens of enzymes. The roles of each enzyme or enzyme class are not fully under-stood, and utilization of a model amenable to genetic analysis should increase thecomprehension of how fungi cope with highly recalcitrant biomass. Here, we reportthat the cellobiose dehydrogenases of the model fungus Podospora anserina enableit to consume crystalline cellulose yet seem to play a minor role on actual sub-strates, such as wood shavings or miscanthus. Analysis of secreted proteins suggeststhat Podospora anserina compensates for the lack of cellobiose dehydrogenase byincreasing beta-glucosidase expression and using an alternate electron donor forLPMO.

KEYWORDS biomass degradation, cellobiose dehydrogenase, Podospora anserina

Cellobiose dehydrogenases (CDHs; EC 1.1.99.18) are extracellular fungal enzymesthat catalyze the oxidation of cellobiose into cellobiono-1,5-lactone (1). CDHs

sometimes harbor one flavin-containing dehydrogenase (FAD-DH) domain from theAA3_1 family, but in most cases both an FAD-DH domain and a cytochrome domainfrom the AA8 family (www.cazy.org) are present (2). FAD-DH oxidizes cellobiose at the

Received 27 September 2016 Accepted 4November 2016

Accepted manuscript posted online 11November 2016

Citation Tangthirasunun N, Navarro D,Garajova S, Chevret D, Tong LCH, Gautier V,Hyde KD, Silar P, Berrin J-G. 2017. Inactivation ofcellobiose dehydrogenases modifies thecellulose degradation mechanism of Podosporaanserina. Appl Environ Microbiol 83:e02716-16.https://doi.org/10.1128/AEM.02716-16.

Editor Daniel Cullen, USDA Forest ProductsLaboratory

Copyright © 2016 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Philippe Silar,[email protected], or Jean-GuyBerrin, [email protected].

N.T. and D.N. contributed equally to this work.

BIODEGRADATION

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C-1 position to cellobiolactone by reduction of FAD, followed by its reoxidation thanksto acceptors such as quinones and phenoxy radicals. CDHs are secreted by a variety offungi, including white-rot, brown-rot, and soft-rot fungi, as well as molds (1), upongrowth on cellulose and plant biomass (3, 4). The first CDHs, initially named cellobioseoxidase, were discovered as enzymes able to catalyze the cellobiose-dependent reduc-tion of quinones (5, 6). Their enzymatic properties have been characterized in manyfungi, including Phanerochaete chrysosporium (7), Trametes versicolor (8), Schizophyllumcommune (9), Pycnoporus cinnabarinus (3), Pycnoporus sanguineus (10), Cerrena unicolor(11), Podospora anserina (12, 13), and Neurospora crassa (14–17). Zámocký et al. (18)divided the CDH family into three phylogenetic branches. Class I consists of CDH frombasidiomycetes and does not contain a family 1 carbohydrate binding module (CBM1),although most CDHs bind to cellulose (14). Class II contains only ascomycete CDHs thatdo (class IIA) or do not (class IIB) harbor a CBM1 module.

CDH appears to be involved in the degradation of both lignin and cellulose by fungi(1, 8, 9, 15, 16, 19, 20). Nevertheless, their actual function is still not fully understood (1,9, 15, 16). Recently, fungal CDHs were shown to act as a reductant for lytic polysac-charide mono-oxygenases (LPMOs), providing electrons for the redox-mediated oxida-tive cleavage of cellulose (14, 17, 21–23). The electrons are rapidly transferred from theflavin to the heme domain of CDH via the heme propionate group (24–26). Followingheme reduction, the cytochrome domain of CDH reduces the copper in the active siteof the LPMO to initiate oxidative cellulose breakdown (27).

Although large amounts of data are available on the biochemistry of these enzymes,little is known about their genetics. In basidiomycetes, a CDH gene has only beeninactivated in T. versicolor (28), and the single CDH of this species was found to beimportant for wood invasion but not for kraft pulp delignification. Recently, Phillips etal. (14) and Zhang et al. (15) analyzed the phenotypes of the deletions of cdh1 and cdh2genes in N. crassa and found a reduction in CDH activity. However, they did notinvestigate thoroughly the effect of the deletion on fungal physiology and on plantbiomass degradation. To gain insight into the actual role of CDH, we performed athorough analysis of the role of the three enzymes encoded by the genome of thefilamentous ascomycete P. anserina. This filamentous fungus is easy to manipulate andis able to rapidly complete its life cycle with wood as the sole carbon source (29–31).Gene deletions and construction of multiple mutants can rapidly be achieved. More-over, genome sequencing (30) has revealed that its genome contains a large numberof genes putatively involved in lignocellulose breakdown. Comparison with other fungirevealed that P. anserina has a large number of such genes, like basidiomycetes (2, 30).Being genetically tractable and having a complement of genes implicated in lignocel-lulose breakdown similar to that of basidiomycetes make it a good model to decipherhow fungi manage to break down lignocellulose. Here, we investigated the in vivo roleof three P. anserina genes encoding CDHs by targeted gene deletion and analyzedphenotypes and secretomes of the mutants.

RESULTS AND DISCUSSIONSequence analyses of the three CDHs of Podospora anserina. As in previous

analyses of the first version of the P. anserina genome sequence (12), analysis of acorrected version of the complete genome of P. anserina (32) revealed the presence ofthree sequences potentially coding for CDHs, Pa_7_2650 or P. anserina CDH1 (PaCDH1)(formerly CDHpa) (12), Pa_0_280 or PaCDH2 (formerly PaCDHB) (13), and Pa_6_11360 orPaCDH3 (Fig. 1). A domain search revealed that PaCDH2 lacked a CBM1 module andthus belonged to class IIB, while PaCDH1 contained a CBM1 module at its C terminusand thus belonged to class IIA. These two CDHs were previously characterized as ableto oxidize cellobiose (12, 13) and to provide electrons to P. anserina AA9 LPMOs forefficient cleavage of cellulose (13). Intriguingly, PaCDH3 does not contain an AA8cytochrome domain and lacks a CBM1 module. Lack of an AA8 cytochrome domain isnot unprecedented, as an active CDH missing this domain thanks to proteolyticcleavage has been purified from T. versicolor (8). SignalP (33) and Predisi (33) predicted

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a secretion signal for PaCDH1 and PaCDH2 but not for PaCDH3. Analysis of a P. anserinaexpressed sequenced tag database (30), transcriptome sequencing (RNA-seq; R. Debu-chy, personal communication), as well as microarray data (34) validated the annotationof the three genes and showed that they were expressed but not regulated duringmycelium growth. However, expression of PaCDH1 was significantly increased in mu-tants of the PaNox1, PaMpk1, and PaMpk2 genes; PaCDH3 expression was significantlyincreased in mutants of PaMpk1 and PaMpk2 genes and PaCDH2 in mutants of thePaMpk2 genes. PaNox1, PaMpk1, and PaMpk2 belong to a signaling pathway involvedin numerous aspects of the P. anserina life cycle, including regulation of biomassdegradation, and microarray analysis of the transcriptomes of these mutant are avail-able (34).

Exploration of the genomes of other filamentous fungi showed that CDH genes werenot present in the available genomes of basal fungi, i.e., Chrytridiomycota, Neocallimas-tigomycota, Blastocladiomycota, Kickxellomycotina, Mucoromycotina, Entomophthoromy-cotina, and Glomeromycota. They were also lacking in the available genomes ofTaphrinomycotina, Saccharomycotina, Pucciniomycotina, and Ustilaginomycotina. More-over, they were not present in all genomes from Pezizomycotina and Agaricomycotina,including some species known to be excellent wood degraders, such as Fomitopsispinicola and Neolentinus lepideus. Phylogenetic analysis performed with a selectedgroup of CDHs (Fig. 2) showed that PaCDH1 was orthologous to Neurospora crassaCDH1, also a class IIA enzyme, and PaCDH2 was orthologous to CDH2, a class IIB one.PaCDH3 has no orthologous gene in N. crassa, which only has two enzymes, andclustered with those from Magnaporthe grisea (Maggr1I113216) and Daldinia eschschol-zii (DalEC12I83623). Unlike PaCDH3, Maggr1I113216 has a predicted AA8 cytochromedomain, while DalEC12I83623 does not. Note that M. grisea has four additional genesencoding putative CDH, and only one of those has the cytochrome binding domainwhile the three others do not. All of the M. grisea CDHs have predicted secretion signals,but DalEC12I83623 does not. Loss/gain of the AA8 domain, associated with loss orretention of the secretion signal, thus seems a frequent event during fungal evolution.

Phenotypic analyses of CDH-deleted strains. To explore the role of PaCDH1,PaCDH2, and PaCDH3, the three genes were inactivated by targeted gene deletion andthe mutants were validated by Southern blotting (see Fig. S1 in the supplementalmaterial). Three strains, PaCDH1Δ, PaCDH2Δ, and PaCDH3Δ, inactivated for PaCDH1,PaCDH2, and PaCDH3, respectively, were obtained. To explore possible functionalredundancy, the PaCDH1Δ PaCDH2Δ, PaCDH1Δ PaCDH3Δ, and PaCDH2Δ PaCDH3Δ doublemutants, as well as the PaCDH1Δ PaCDH2Δ PaCDH3Δ (CDHΔ) triple mutant lacking all ofthe CDH genes, were constructed by genetic crosses thanks to the different selectionmarkers used to inactivate each gene. A thorough phenotypic analysis then wasperformed and compared to the wild type and the CATΔΔΔΔΔ mutant inactivated for allfive catalase genes present in the P. anserina genome (35). Growth and fertility of thestrains were first evaluated on M2, the minimal medium used to assess P. anserinadevelopment. Because formation of fruiting bodies requires energy, assessing not only

FIG 1 Modular organization of Podospora anserina CDHs. Domain organization of the three P. anserinaCDHs. AA8, cytochrome domain from the AA8 family; FAD, FAD-DH domain; CBM1, carbohydrate bindingmodule 1.

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growth but also fertility enabled us to assess whether the mutants were as proficientas the wild type at retrieving nutrients from the offered food sources. The M2 mediumcontains dextrin as the sole carbon source. As seen in Fig. 3A, the growth rate was notaltered in any of the CDH mutants on this medium; fertility of all of the mutants,including germination efficiency of the ascospores, also was not modified in thismedium (Fig. 3B).

Growth and fertility were then assayed on media containing various carbon sources,including glucose, crystalline and fibrous cellulose, alkali-sulfonated lignin, Guibourtiademeusi wood shavings, and shredded miscanthus. On glucose medium, growth ofwild-type P. anserina is slowed and fertility severely diminished. As seen in Fig. S2, nomodification of growth rate was observed on glucose medium in any of the CDHmutants, and their fertility, but not the germination efficiency of the ascospores, wasdiminished as described for the wild type. On wood shavings and miscanthus, we didnot detect any obvious phenotype (Fig. S3), suggesting that CDH is dispensable forscavenging nutrients from these carbon sources. This is different from the results ofDumonceaux et al. (28), who observed an inability to grow on wood. Unfortunately,Southern blot analysis did not provide full confirmation that inactivation of the singleCDH gene was responsible for the phenotype in the Dumonceaux et al. paper, leavingthe possibility that lack of growth on wood was due to an additional genetic defect.Alternatively, the difference may be due to the fact that ascomycetes and basidiomy-cetes rely on different levels of (compensated or uncompensated) CDH activity. More-over, because both wood shavings and miscanthus are very heterogeneous, growthrates, which can be different from the plate assay ones, cannot be measured, and smalldifferences in fertility are somewhat problematic to evaluate correctly. Similarly, growthrate was not modified on alkali-sulfonated lignin, unlike that of the CATΔΔΔΔΔ mutant,which was decreased as described previously (Fig. S3) (35). When assayed with paper

FIG 2 Phylogenetic tree of selected fungal CDHs. The Podospora anserina CDHs are boxed.

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which contained fibrous cellulose, fertility of all mutants also was not affected (Fig. S4).Lack of effect of the mutation on the ability to degrade paper was confirmed byevaluating the weight loss of paper pads, as we could not detect any statisticallysignificant differences between the wild type and the analyzed mutants (Fig. S4). Onthe contrary, and interestingly, when assayed with crystalline cellulose, fertility asmeasured by the number of perithecia was diminished by a factor of two specifically forPaCDH1Δ PaCDH2Δ and CDHΔ, while germination efficiency, growth rate, and myceliumdensity were not altered (Fig. 4). Owing to the lack of phenotype of the single mutants,we could not easily engage complementation experiments. Thus, we could not com-pletely rule out that this phenotype was due to an additional unintended geneticdefect. Nevertheless, the phenotype was observed in three independently constructeddouble mutants and three independently constructed triple mutants. Unaltered growth

FIG 3 Growth rate (A) and fertility (B) of the wild type and CDH mutants on M2 containing dextrins asa carbon source. (A) Diameters of cultures 3 and 5 days after inoculation. Data are averages from threecultures. (B) On this medium, fruiting bodies are formed along a ring that centers on the inoculationpoint. The fruiting bodies (inset on the wild type) are the small black dots.

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rate and mycelium density indicated that these mutants could not retrieve morenutrients from crystalline cellulose. Thus, the lower fertility of these mutants was dueto impairment in their ability to scavenge nutrients from crystalline cellulose. Overall,we could not detect such dramatic effects as those created by deleting catalases (Fig.S3) (35) or laccases (36, 37), suggesting either a minor role of CDH in degradation ofcomplex biomass or that P. anserina has compensatory mechanisms that enable it tocope with a lack of CDH. It is known from previous work that many compounds orproteins may be used as electron donors by LPMOs (38–40). Nonetheless, CDH activity

FIG 4 Growth rate (A) and fertility (B) of the wild type and CDH mutants on M4 containing crystallinecellulose as the sole carbon source. (A) Diameters of cultures 3 and 5 days after inoculation. Data areaverages from three cultures. (B) On this medium, fruiting bodies are also formed along a ring thatcenters on the inoculation point. However, this ring is larger than that on M2. It may vary from oneexperiment to the other, owing to the heterogeneous nature of the medium. Nevertheless, PaCDH1Δ

PaCDH2Δ (CDH1Δ CDH2Δ) and CDHΔ (CDH1Δ CDH2Δ CDH3Δ) exhibited a constant diminution of fertility, asseen by the estimated number of perithecia (indicated at the bottom of each petri plate).

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is important in P. anserina for the degradation of crystalline cellulose, as seen by thelower fertility of the PaCDH1Δ PaCDH2Δ and CDHΔ mutants when grown on this foodsource. Crystalline cellulose is present at various levels in the different biomasses eatenby herbivores, i.e., the growth substrates of this coprophilous fungus. Therefore, smalldecreases in growth and fertility of strains lacking CDH, not detected here in the woodshaving/miscanthus experiments because of their heterogeneous nature or becausecrystalline cellulose represents only a minor fraction of the total cellulose, could in thewild and in the long term have a large impact on fitness.

Because CDH may produce peroxide as a final product, we assayed production ofperoxide and superoxide by wild-type and CDH mutant thalli using the diaminoben-zidine (DAB) and nitroblue tetrazolium assays (NBT), respectively (41). We could notdetect any differences among the mutants in the production of both peroxide andsuperoxide, as previously seen for the CATΔΔΔΔΔ mutant (Fig. S5) (35). This suggestedthat CDHs were minor sources of reactive oxygen species (ROS) in P. anserina; likewise,catalase had a minor role in removing peroxide under the conditions used for theassays, as previously hypothesized (35). We also assayed with the DAB assay theproduction of ROS when P. anserina encounters Penicillium chrysogenum. At the contactpoint, P. anserina exerts hyphal interference and kills P. chrysogenum hyphae (42). Asseen in Fig. S5, this process was not modified in the CDH mutants. Overall, it thusappears that CDHs have a minor role in peroxide/superoxide production in P. anserina,as detected here by the DAB and NBT assays.

Proteomic analyses of Podospora anserina secretomes. Analyses of wild-type P.

anserina and the CDHΔ triple mutant was performed using a secretomic analysisapproach. P. anserina was grown on lignocellulosic biomass (i.e., wheat straw) to favorthe secretion of lignocellulose-acting enzymes. Enzymatic assays showed that the CDHactivity was equal to 0.429 nkat/ml in the wild type and null in CDHΔ. Liquidchromatography-tandem mass spectrometry (LC-MS/MS) analyses of secretomes al-lowed the identification of about 50 secreted proteins (Table 1). Surprisingly, the mostabundant protein in both secretomes was a putative tannase/esterase (Pa_5_7020;GenBank accession number CAP65121). Growth of both P. anserina wild-type and CDHΔ

strains on wheat straw favored the secretion of lignocellulose-acting enzymes (e.g., AA1and AA5) and CAZymes targeting plant cell wall components (e.g., GH3, GH6, GH7, andGH74). Among them, five harbored a CBM1 module, i.e., a GH7-CBM1 putative cello-biohydrolase (Pa_3_730; CAP61105), a putative GH74-CBM1 xyloglucanase (Pa_4_7820;CAP66717), a GH6-CBM1 cellobiohydrolase (Pa_0_1250; CAP60942) (43), a GH131-CBM1broad-specificity glucanase (Pa_3_10940; CAP61309) (44), and, in the wild type, PaCDH1(Pa_7_2650; CAP68427) (12). As expected, PaCDH1 was absent from the secretome ofthe CDHΔ mutant strain (Table 1). The main striking difference between the wild-typeand the CDHΔ mutant secretomes concerned the GH3 family, which consists mainly ofputative �-glucosidases. The CDHΔ mutant secretome contained five different GH3enzymes (CAP61089, CAP64656, CAP68372, CAP73569, and CAP60165), while the wild-type secretomes only contained the most abundant one (CAP61089) (Table 1). Thisobservation could be explained by the fact that expression of �-glucosidases is inducedby cellobiose, as observed in the related fungus Neurospora crassa (45). Therefore, CDHcould participate in the regulation of the expression of �-glucosidases in P. anserina.Moreover, it has been suggested that CDH could enhance cellulase activity by relievingproduct inhibition of cellobiohydrolases through the oxidation of cellobiose to cello-bionic acid (46). P. anserina may compensate for the lack of CDH with the secretion of�-glucosidases to favor GH6 and GH7 cellobiohydrolase activity by relieving productinhibition. This mechanism might in part explain the weak phenotypes of the CDHdeletion mutants.

Cellulose degradation potential of Podospora anserina secretomes. To furtherevaluate the importance of CDH in the degradation of cellulose, wild-type and mutantsecretomes were tested for their capacity to degrade cellulose. Both secretomes wereable to efficiently convert cellulose into soluble products yielding significant amounts

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TABLE 1 Comparative analysis of the CAZymes identified in the wild-type and CDH�

mutant secretomesa

GenBankaccessionno. Functional annotation

CAZyannotation

Total no. of spectra

Wild-typestrain

MutantCDH�strain

CAP65121.1 Tannase and feruloyl esterase(pfam07519)

152 230

CAP61636.1 Putative aromatic peroxygenase 38 14CAP68535.1 Catalase 32 35CAP61105.1 Cellobiohydrolase GH7-CBM1 48 12CAP61378.1 FAD oxidoreductase 44 11CAP68327.1 Beta-1,3-glucanase GH55 14 32CAP64719.1 Putative laccase AA1_3 27 9CAP61089.1 Beta-glucosidase GH3 23 14CAP60942.1 Cellobiohydrolase GH6-CBM1 30 4CAP66717.1 Xyloglucanase GH74-CBM1 22 10CAP73016.1 �-1,3-Glucanosyltransglycosylase GH72-CBM43 27 3CAP61475.1 Carboxyl ester hydrolase 14 9CAP70294.1 Glucoamylase GH15-CBM20 7CAP61309.1 Beta-glucanase GH131-CBM1 13 2CAP60434.1 Copper radical oxidase AA5_1 14CAP66761.1 Glycanase GH16 13 2CAP65628.1 Copper oxidoreductase 13CAP64656.1 Beta-glucosidase GH3 8CAP68372.1 Beta-glucosidase GH3 5CAP59879.1 �,�-Trehalase GH37 5 6CAP61625.1 Unknown function 5 2CAP66532.1 Beta-1,3-glucanase GH55 10CAP61276.1 Unknown function 8 2CAP65464.1 FAD oxidoreductase 3CAP68427.1 Cellobiose dehydrogenase (PaCDH1) AA3_1-AA8 9CAP62084.1 FAD oxidoreductase 8CAP70307.1 Beta-glucanase GH17 8CAP68194.1 Carboxyl ester hydrolase 8CAP65285.1 GMC oxidoreductase AA3_2 6CAP61511.1 Peptidase s28 family 5CAP65657.1 Beta-glucanase GH17 3 3CAP61726.1 Putative glutaminase (pfam08760) 4 3CAP60040.1 Unknown function 5 2CAP73527.1 Peptidase a4 2 2CAP73079.1 Unknown function 2 3CAP65694.1 Unknown function 3CAP73947.1 Unknown function 4 2CAP66439.1 Putative dehydrogenase 6CAP65311.1 Unknown function 5CAP68130.1 Chitinase GH18-CBM50 2CAP71075.1 Unknown function 2 3CAP68366.1 Unknown function 2CAP67351.1 Unknown function 3CAP61335.1 FAD oxidoreductase 2 3CAP61538.1 Unknown function 2 2CAP68226.1 Protease s28 3CAP73569.1 Beta-glucosidase GH3 4CAP70690.1 Putative aldose 1-epimerase 4CAP72727.1 Glycanase GH16 2 2CAP61110.1 Unknown function 2CAP70412.1 Glucoamylase GH15-CBM20 3CAP70942.1 Aspartic protease 3CAP60165.1 Beta-glucosidase GH3 3CAP64871.1 GMC oxidoreductase AA3 3CAP73573.1 Unknown function 2CAP65364.1 Unknown function 2CAP62325.1 GMC oxidoreductase AA3_2 2CAP67022.1 FAD oxidoreductase 2aProteins are listed according to their putative function and CAZy annotation. Total spectra of uniquepeptide sequences identified by LC-MS/MS in each secretome are indicated.

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of glucose (DP1) and cellobiose (DP2). Glucose yields were more than 2-fold higher inthe case of the CDHΔ mutant secretome (10.0 �M) compared to the WT secretome (4.24�M) (Fig. 5A). Addition of the AA9 LPMO from P. anserina (PaLPMO9H) (13) boosted theconversion of cellulose in both cases (wild-type and CDHΔ mutant secretomes). Reac-tion mixtures contained oxidized cello-oligosaccharide products (C-4- and C-1/C-4-oxidized products), in agreement with the regioselectivity of PaLPMO9H (Fig. 5B). As noexternal electron donor was added to the enzymatic reaction, these results provideevidence that redox partners other than CDH are present in the CDHΔ mutant secre-tomes, triggering oxidative cleavage of cellulose by PaLPMO9H. One can suggest gallicor tannic acids as a potential candidate. Indeed, these acids have been shown to bealternative electron donors for LPMOs (40), and the putative tannase/esterase thatcould be involved in the production of these molecules is present in the mutantsecretome. We have thus tested the ability of tannic acid to restore a higher fertility tothe CDHΔ mutant by adding various quantities of this product to M4 plates. As apositive control we used ascorbate, a universal electron donor, and as a negativecontrol we used dehydroascorbate (D-ascorbate), the oxidized form of ascorbate. As

FIG 5 Enzymatic degradation of cellulose using fungal secretomes. Chromatograms highlight solubleproducts identified after enzymatic treatment with wild-type (WT) and CDH triple mutant (CDHΔ)secretomes alone (A) and secretomes supplemented with PaLPMO9H (B). (A) WT, CDHΔ, and PASCrepresent control conditions. The inset highlights the identification of minor oxidized and nonoxidizedproducts. (B) Oxidized oligosaccharides are identified only under conditions where PaLPMO9H isincubated with either WT or CDHΔ mutant secretome. Oxidized cello-oligosaccharides were identifiedbased on previous analysis (14).

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expected, ascorbate promoted a restoration of CDHΔ fertility in a dose-dependentmanner, while D-ascorbate did not (Fig. S6). Tannic acid was not able to restore fertilityof CDHΔ (Fig. S6), suggesting that it may not be the electron donor compensating fora lack of CDH on complex biomass. It was even toxic at high doses for P. anserina (Fig.S6, compare the fertility of the wild-type cross at 0.02 g/liter of tannic acid with that atlower concentrations). The genome of P. anserina encodes many other potentialcandidates that could act as electron donors (30). For example, the flavo-oxidase fromthe AA3_2 subfamily (Pa_5_5180; CAP65285) identified in the CDHΔ mutant secretome(Table 1) could provide electrons to LPMOs, therefore triggering oxidative cellulosedegradation. Phylogenetic prediction of the function of this flavo-oxidase is hamperedby the small number of characterized members of the AA3_2 subfamily (47). Ourfindings are in agreement with recent findings showing that AA3_2 GMC oxidoreduc-tases can serve as extracellular electron sources for LPMOs (38, 39), thus extending thearray of fungal redox partners in filamentous fungi. Overall, the present data suggestthat compensatory mechanisms indeed enable P. anserina to cope with lack of CDH forthe most part.

Conclusions. Analysis by targeted gene deletion of the CDH genes of P. anserina hasshown that these enzymes are important for degradation of crystalline cellulose, butthat their absence does not result in a dramatic decrease in the ability to use complexbiomasses, such as wood shavings or miscanthus, in which crystalline cellulose mayrepresent a minor fraction. The efficiency of the mutant CDHΔ secretome to degradecellulose appeared similar to that of the wild type, except that twice as much cellobiosewas obtained as expected from the lack of conversion by CDH. Analysis of oxidizedproducts indicated that LPMOs are still active, suggesting that additional partners ableto provide electrons to LPMOs are present in CDHΔ. Based on its increased quantity inthe CDHΔ secretome, the AA3_2 flavo-oxidase Pa_5_5180 (CAP65285) may be a goodcandidate for such a task. These data underscore the complex mechanisms of plantbiomass degradation by fungi in which many activities, some potentially redundant, actin concert to achieve efficient degradation. This is particularly important for coprophi-lous fungi, such as P. anserina, because they likely encounter biomasses of diverseorigins, which have been more or less extensively digested by herbivores, but also bythe other competing fungi that grow and fructify in succession on dung.

MATERIALS AND METHODSStrains and growth conditions. The strains used in this study derived from the S wild-type strain

(48) used for sequencing (30, 32). Standard culture conditions, media, and genetic methods for P.anserina have been described (29, 49). The M2 medium has the following composition: 0.25 g/literKH2PO4, 0.3 g/liter K2HPO4, 0.25 g/liter MgSO4·7H2O, 0.5 g/liter urea, 0.05 mg/liter thiamine, 0.25�g/liter biotine, 2.5 mg/liter citric acid, 2.5 mg/liter ZnSO4, 0.5 mg/liter CuSO4, 125 �g/liter MnSO4,25 �g/liter boric acid, 25 �g/liter sodium molybdate, 25 �g/liter iron alum, dextrine 5 g/liter, 12.5g/liter agar. M4 medium has the same composition except that dextrin is replace by crystallinecellulose (microgranular CC31 cellulose powder; catalog no. 401050; Whatman). M0 has the samecomposition as M2 except that it lacks dextrin, which can be replace by the same amount/weightof autoclaved alkali-sulfonated lignin, wood shavings, or shredded miscanthus. Alkali-sulfonatedlignin was purchased from Sigma-Aldrich (catalog no. 471003). Perithecium numbers were estimatedusing dotcount (http://reuter.mit.edu/software/dotcount/).

Gene deletions and phenotypic analysis. The PaCDH1, PaCDH2, and PaCDH3 genes were deletedas described in reference 29 using resistance markers to Geneticin (50), hygromycin B (51), andphleomycin (51), respectively. After verification by Southern blotting (see Fig. S1 in the supplementalmaterial), one verified mat� and one verified mat-deleted strain were picked for phenotypic analyses.Double and triple mutants were constructed by genetic crosses, and genotypes were ascertained thanksto the resistance markers. At least three independently constructed PaCDH1Δ PaCDH2Δ and CDHΔ

mutants were analyzed for fertility on M4. All presented the fertility defect. The primers used aredescribed in Table S1. Phenotypes were analyzed as described previously for fertility on various carbonsources (35–37), for paper degradation (52), for constitutive peroxide and superoxide production (41),and for hyphal interference (42).

Phylogenetic analysis. CDH genes of P. anserina were identified by BLAST using various fungal CDHsas the query. Alignment was made with MAFFT (53) and manually refined. This alignment was used toconstruct a phylogenetic tree using the maximum likelihood method (PhyML software) (54) andtransferred to the iTOL server for visualization (55). Bootstrap values are expressed as percentages of 100replicates. Signal peptides were predicted using SignalP 4.1 at http://www.cbs.dtu.dk/services/SignalP/and Predisi at http://www.predisi.de.

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Secretome preparation and CDH activity assay. On the basis of previous studies, the fungalcultures were grown in a liquid medium containing 15 g liter�1 (based on the dry matter) wheat straw(Triticum aestivum Apache, France) as a carbon source, 2.5 g liter�1 maltose as a starter, 1.842 g liter�1

diammonium tartrate, 0.5 g liter�1 yeast extract, 0.2 g liter�1 KH2PO4, 0.0132 g liter�1 CaCl2, and 0.5 gliter�1 MgSO4. Fungal cultures were prepared in biological triplicate using 200-ml baffled flasks contain-ing 100 ml of culture medium. Cultures were inoculated with mycelial fragments from five fungal disks(4-mm diameter) crushed in 1 ml minimum medium, using a FastPrep-24 system (MP Biomedicals, Illkirch,France) set to 40 m s�1 for 60 s. Liquid cultures were incubated at 25°C with orbital shaking at 140 rpm(Infors HT, Switzerland). All of the cultures were stopped at 4 days after inoculation. The culture broths(secretomes) were harvested and pooled (total volume, 250 to 300 ml), filtered (using a 0.2-�m-pore-sizepolyethersulfone membrane; Vivaspin; Sartorius, Germany), diafiltered, and concentrated (Vivaspin poly-ethersulfone membrane with a 10-kDa cutoff; Sartorius) in 50 mM acetate solution buffer, pH 5, to a finalvolume of 5 ml and then stored in aliquots at �20°C until use. The total amount of protein was assessedusing Bradford assays (protein assay dye reagent concentrate; Bio-Rad, Ivry, France) with a bovine serumalbumin (BSA) standard that ranged from 0.2 to 1 mg ml�1. CDH enzymatic activity was assayed asdescribed in reference 13.

Saccharification assays. The concentrated secretomes were tested for their ability to hydrolyzephosphoric acid-swollen cellulose (PASC) suspension (0.1%, wt/vol) prepared in 50 mM acetate buffer, pH5. The enzyme reactions were performed in 2-ml tubes and incubated in a thermomixer (Eppendorf,Montesson, France) at 50°C and 850 rpm. After 24 h of incubation, all of the samples were boiled at 100°Cfor 10 min to stop the enzymatic reaction and then centrifuged at 16,000 rpm for 15 min at 4°C toseparate the soluble fraction from the remaining insoluble fraction before carbohydrate determination.Assays were performed as triplicate independent experiments. The reaction mixture (300 �l liquidvolume) contained 3 �g of total protein of P. anserina secretome. For supplementation of secretomeswith recombinant P. anserina LPMO, 2 �M purified recombinant PaLPMO9H (13) was added. Assays wereperformed as triplicate independent experiments.

Analysis of oxidized and nonoxidized oligosaccharides. Monosaccharides, oligosaccharides, andtheir corresponding aldonic acid forms generated after PASC and Avicel cleavage were analyzed by ionicchromatography (high-performance anion-exchange chromatography) as described by Bennati-Granieret al. (13), using nonoxidized oligosaccharides (Megazyme) as standards. Corresponding C-1-oxidizedstandards (from DP2 to DP6) were produced from nonoxidized cello-oligosaccharides by CDH treatmentas described by Bennati-Granier et al. (13). All assays were carried out in triplicate.

Proteomic analysis of secretomes. Short SDS-PAGE runs (precast 4 to 12% Bis-Tris mini gels;Invitrogen, France) were performed, allowing proteins diafiltered from pooled biological triplicates ofsecretomes (10 �g) to migrate to a length of 0.5 cm, and gels were stained with Coomassie blue (Bio-Rad,Marnes-la-Coquette, France). Each one-dimensional electrophoresis lane was cut into two slices of gel (2mm in width), and protein identification was performed using PAPPSO (Plateforme d’Analyze Pro-téomique de Paris Sud-Ouest) platform facilities. In-gel digestion was carried out according to a standardtrypsinolysis protocol. Gel pieces were washed twice with 50% (vol/vol) acetonitrile (ACN), 25 mMNH4CO3 and incubated in the presence of 10 mM dithiothreitol (DTT) for 1 h at 56°C. After cooling, thesupernatant was removed and the samples were incubated with 55 mM iodoacetamide at roomtemperature in the dark. Gel plugs were washed with ACN and then dried in a vacuum speedconcentrator. Digestion was performed for 8 h at 37°C with 200 ng of modified trypsin (Promega,Charbonnières-les-Bains, France) dissolved in 25 mM NH4CO3. Tryptic peptides were first extracted with50% (vol/vol) CAN and 0.5% (vol/vol) trifluoroacetic acid (TFA) and then with pure ACN. Peptide extractswere dried in a vacuum speed concentrator (Thermo Fisher Scientific, Villebon sur Yvette, France) andsuspended in 25 �l of 2% (vol/vol) ACN, 0.05% (vol/vol) TFA, and 0.08% (vol/vol) formic acid. High-performance liquid chromatography (HPLC) was performed on a NanoLC-Ultra system (Eksigent, Les Ulis,France). Trypsin digestion products were first concentrated and desalted on a precolumn cartridge(PepMap 100 C18; 0.3 by 5 mm; Dionex, Thermo Fisher Scientific) with 0.1% HCOOH at 7.5 �l min�1 for3 min. The precolumn cartridge was connected to the separating column (C18; 0.075 by 0.15 mm;Biosphere Nanoseparations, Nieuwkoop, The Netherlands), and the peptides were eluted with a lineargradient from 5 to 35% ACN in 0.1% HCOOH for 40 min at 300 nl min�1. On-line analysis of peptides wasperformed with a Q-exactive mass spectrometer (Thermo Fisher Scientific, USA), using a nanoelectros-pray ion source. Ionization (1.8-kV ionization potential) was performed with a stainless steel emitter(30-�m inner diameter; Thermo Electron, Villebon sur Yvette, France). Peptide ions were analyzed usingXcalibur 2.1 (Thermo Scientific, Villebon sur Yvette, France) with the following data-dependent acquisi-tion steps: step 1, full MS scan (mass-to-charge ratio [m/z], 400 to 1,400; resolution, 70,000); step 2, MS/MS(normalized collision energy, 30%; resolution, 17,500). Step 2 was repeated for the eight major ionsdetected in step 1. Dynamic exclusion was set to 40 s. The raw mass data were first converted to mzXMLformat with the ReAdW software (SPC Proteomics Tools, Seattle, WA). Protein identification was per-formed by querying MS/MS data against databases, together with an in-house contaminant database,using the X!Tandem software (X!Tandem Cyclone, Jouy en Josas, France) with the following parameters:one missed trypsin cleavage allowed, alkylation of cysteine and conditional oxidation of methionine, andprecursor and fragment ion set at 2 ppm and 0.005 Da, respectively. A refined search was added withsimilar parameters, except that semitryptic peptides, possible N-terminal acetylation, and histidinemono- and dimethylations were searched. All peptides matched with an E value lower than 0.05 wereparsed with X!Tandem pipeline software. Proteins identified with at least two unique peptides and alog(E value) of lower than �2.6 were validated.

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://dx.doi.org/10.1128/AEM.02716-16.

TEXT S1, PDF file, 4.5 MB.

ACKNOWLEDGMENTSThis work was supported by Ambassade de France à Bangkok, intramural funding

from Universités Paris 7 and Paris 11, and grant P3AMB from Region Ile de France.We thank Sylvie Cangemi for expert technical assistance.

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