# Identification of Copper-Containing Oxidoreductases in the Secretomes of Three Colletotrichum Species with a Focus on Copper Radical Oxidases for the Biocatalytic Production of Fatty Aldehydes

David Ribeaucourt, Safwan Saker, David Navarro, Bastien Bissaro, Elodie Drula, Lydie Oliveira Correia, Mireille Haon, Sacha Grisel, Nicolas Lapalu, Bernard Henrissat, et al.

► To cite this version:

David Ribeaucourt, Safwan Saker, David Navarro, Bastien Bissaro, Elodie Drula, et al. Identification of Copper-Containing Oxidoreductases in the Secretomes of Three Colletotrichum Species with a Focus on Copper Radical Oxidases for the Biocatalytic Production of Fatty Aldehydes. Applied and Environmental Microbiology, 2021, 87 (24), pp.e01526-21. 10.1128/AEM.01526-21. hal-03619439

HAL Id: hal-03619439

https://hal.science/hal-03619439

Submitted on 5 May 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Identification of copper-containing oxidoreductases in the secretomes of three

2 *Colletotrichum* species with a focus on copper radical oxidases for the

3 biocatalytic production of fatty aldehydes

4 David Ribeaucourt<sup>a,b,c</sup>, Safwan Saker<sup>a,1</sup>, David Navarro<sup>a</sup>, Bastien Bissaro<sup>a</sup>, Elodie Drula<sup>a,d,e</sup>,

5 Lydie Oliveira Correia<sup>f</sup>, Mireille Haon<sup>a</sup>, Sacha Grisel<sup>a</sup>, Nicolas Lapalu<sup>g</sup>, Bernard Henrissat<sup>d,e,h</sup>,

6 Richard J. O'Connell<sup>g</sup>, Fanny Lambert<sup>c</sup>, Mickaël Lafond<sup>b,#</sup> and Jean-Guy Berrin<sup>a,#</sup>

7 <sup>a</sup>INRAE, Aix Marseille Univ, UMR1163 Biodiversité et Biotechnologie Fongiques, Marseille,

8 France

9 <sup>b</sup>Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France

10 <sup>c</sup>V. Mane Fils, 620 route de Grasse, Le Bar sur Loup, France

11 <sup>d</sup>INRAE, USC1408, AFMB, Marseille, France

12 <sup>e</sup>CNRS, Aix Marseille Univ, UMR7257, AFMB, Marseille, France

13 <sup>f</sup>Plateforme d'Analyse Protéomique de Paris Sud-Ouest, INRAE, AgroParisTech, Université

14 Paris-Saclay, Micalis Institute, Jouy-en-Josas, France

15 <sup>g</sup>UMR BIOGER, Institut National de la Recherche Agronomique, AgroParisTech, Université

16 Paris-11Saclay, Versailles, France

17 <sup>h</sup>Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

18 <sup>i</sup>corresponding authors:

19 Jean-Guy Berrin (<u>jean-guy.berrin@inrae.fr</u>)

20 Mickaël Lafond (<u>mickael.lafond@univ-amu.fr</u>)

21

22 Running title: Fungal secretomics and copper radical oxidases

23

<footnote n="1"><sup>1</sup> present address: CRITT BOIS, Epinal, France</footnote>
24 **ABSTRACT**

Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among
fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. With the
initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we
undertook time-course analyses of the secretomes of three *Colletotrichum* species (*C. graminicola*, *C. tabacum* and *C. destructivum*) using proteomics. The addition of a copper-
manganese-ethanol mixture in absence of any plant-biomass mimicking compounds to
*Colletotrichum* cultures unexpectedly induced the secretion of up to 400 proteins, 29-52% of
which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-
containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7,
AA9, AA11-AA13, AA16). Under these specific conditions, while a CRO-glyoxal oxidase from
the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2
CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more
promising strategy for CRO-AlcOx production and utilization. *C. tabacum* and *C. destructivum*
CRO-AlcOx were thus expressed in *Pichia pastoris* and their preference toward both aromatic
and aliphatic primary alcohols was assessed. The CRO-AlcOx from *C. destructivum* was further
investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their
corresponding fragrant aldehydes.

42
43 **IMPORTANCE**

In the context of the industrial shift toward greener processes, the biocatalytic production of
aldehydes is of utmost interest owing to their importance for their use as flavors and fragrances
ingredients. CRO-AlcOx have the potential to become platform enzymes for the oxidation of
alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial application to alleviate various costs pertaining to biocatalysts production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative-stressful conditions we probed, *Colletotrichum* species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, LPMOs) usually assigned to “plant-cell wall degradation”, despite the absence of any plant-biomass mimicking compound, and only little amount of CRO-AlcOx were secreted, pointing out at recombinant expression as the most promising path for their biocatalytic application.

**KEYWORDS:** Copper radical oxidase; CAZymes; Copper-containing enzymes; Alcohol oxidase; Filamentous fungi; Phytopathogen; *Colletotrichum*; Secretomics; Flavors and fragrances.

## INTRODUCTION

The development of a new technological paradigm to support the shift from a fossil-based economy to a greener and more circular economy is at the heart of today's bioeconomic challenges (1). Biotechnology is one of the main levers in this ongoing industrial transformation (2). The development of biotechnology largely relies on biocatalysis and thus require the discovery, control and engineering of a variety of enzymes and microorganisms (3). The fungal kingdom represents a vast and untapped reservoir of biocatalysts (4) but to date, the enzymatic potential of filamentous fungi has mostly been studied in the frame of plant cell wall degradation (5-7). Fungal secretomics is a powerful approach to study the wide diversity of enzymes secreted and to formulate enzymatic cocktails with biotechnological relevance (8-10). Using
suitable inducers, some fungal strains can secrete high amounts of oxidoreductases (11)
including copper-containing enzymes. For instance, hypersecretory fungal strains from the
*Pycnoporus* genus have been selected to produce laccases and use them in applied settings (12,
13).

Most of the copper-dependent enzymes are classified within the Auxiliary Activities (AA) class (14) in the CAZy database (www.cazy.org) (15). The founding member of copper radical oxidases (CROs), a galactose oxidase (*FgrGalOx* – EC 1.1.3.9), was discovered in 1959 from the secretome of *Fusarium graminearum* (16, 17). In 2013, fungal CROs have been classified within the AA5 family and further classified into subfamilies AA5_1 and AA5_2. AA5_1 contains glyoxal oxidases (GLOX - EC 1.2.3.15) that catalyze the oxidation of aldehydes to carboxylic acids, and AA5_2 gathers enzymes that oxidize the alcohol function of diverse substrates (e.g., galactose, galactose-containing polysaccharides, aromatic and aliphatic primary alcohols) to generate the corresponding aldehydes. More recently, the AA5_2 subfamily was shown to be more diverse than expected, encompassing notably various alcohol oxidases (CRO-AlcOx; EC 1.1.3.13) originating from the phytopathogen ascomycete fungi *Colletotrichum* and *Magnaporthe* spp. (18–20). CRO-AlcOx are monocopper, and otherwise organic co-factor free enzymes that catalyze the oxidation of alcohols into aldehydes (with the concomitant reduction of O₂ to H₂O₂). They offer appealing perspectives to produce aldehydes (21), which are indispensable intermediates in many chemical pathways (22, 23) and valuable ingredients for the flavor and fragrance industries (24, 25). In particular, saturated long-chain aldehydes (fatty aldehydes) are widely used for their citrus scent (26, 27), but are currently derived from fossil-based chemistry (28, 29) or extracted from natural plant materials, the supply of which is
93

threatened by emerging diseases (30, 31). Therefore, alternative green and/or natural production

94

pathways must be investigated (32).

95

To date, only pure recombinant forms of CRO-AlcOx have been harnessed in biocatalytic

96

applications (18, 19, 21, 33) and no information on their secretion by natural fungal strains is

97

available. Determining whether CRO-AlcOx encoding fungal strains can secrete these enzymes

98

under laboratory conditions is of strong interest for enzyme selection and technological

99

applications. Noteworthy, aside from the scope of pathogenesis (34–36), very few studies

100

investigated the enzymatic biotechnological potential of *Colletotrichum* spp. (37–40). The use of

101

natural fungal strains could be advantageous to identify and then use potential naturally secreted

102

enzyme partners. In particular, catalase and peroxidase are both required to sustain *in vitro* the

103

activity of isolated CRO-AlcOx and other AA5 (21, 33, 41, 42). Catalase acts as a H₂O₂

104

scavenger while peroxidase is required for CRO-AlcOx activation (20, 33).

105

In this context, we investigated the ability of several fungal strains from the

106

*Colletotrichum* genus to secrete CRO-AlcOx. While pursuing this primary goal, we observed the

107

unexpected secretion of a large panel of plant-biomass degrading oxidoreductases and chitin-

108

related enzymes under oxidative stress conditions and in absence of any plant-cell wall inducers.

109

We report here an integrated study, starting from fungal time-course secretomics, analysis of

110

copper-containing oxidoreductases secretion profiles, with a final focus on the recombinant

111

CRO-AlcOx production and characterization, and biocatalytic application of these enzymes to

112

produce fatty aldehydes for the flavor and fragrance industries.

113

114

## RESULTS

115

### Selection of fungal strains and time-course secretomic study
To investigate the secretion of copper-containing enzymes including AA5_2 CRO-AlcOx by natural fungal strains and their potential use for bioconversion of fatty alcohols to fragrant aldehydes, we selected three fungal phytopathogens from the *Colletotrichum* genus for which genomic information is available. The first obvious choice was *C. graminicola* because the three AA5_2 it encodes have all been previously biochemically characterized (18, 43, 44), one of them being the first, most studied, and to date, most active CRO-AlcOx, namely *CgrAlcOx* (18, 21, 33). By performing a BLAST analysis of the *CgrAlcOx* sequence against the genomes of some other *Colletotrichum* species we had access to, we identified two genes encoding AA5_2 in both *C. tabacum* and *C. destructivum*. For each strain, one gene encoded a putative AlcOx and the other one a putative aryl alcohol oxidase (AAO – based on the recent discovery of an AAO in the AA5_2 family (44)) (Figure S1). These enzymes will thereafter be named *CtaAlcOx/CdeAlcOx* and *CtaAAO/CdeAAO* for *C. tabacum* and *C. destructivum*, respectively. Based on the few studies reporting the detection in some Ascomycota fungal secretomes of a range of copper-containing enzymes from the AA class including the *FgrGalOx* and other AA5 (45–47), we identified copper, manganese and ethanol as possible inducers of AA5. Copper is known to be crucial to activate the *FgrGalOx* and was used previously in combination with manganese to recover active *FgrGalOx* from fungal cultures (47). Ethanol is often used as an inducer of laccases (12) (other copper-containing AAs), and we noticed the presence of CROs in some secretomes induced with ethanol (45). We therefore elaborated two liquid growth media: (i) “YG” (for “Yeast-extract, Glucose”), and (ii) ‘YG-CME” (for “YG-Copper, Ethanol, Manganese”), with the same composition as YG but supplemented with CuSO4, manganese and ethanol. A time-course analysis was performed on each strain in both media at three time points (day 3, day 7 and day 14) and proteomic analyses were performed on the 16 secretomes
generated (note that no samples were harvested at day 3 for C. graminicola in both YG and YG-CME due to insufficient growth).

The quantification of total proteins revealed an increasing amount of proteins over time (except for C. destructivum) (Figure S2) while capillary electrophoresis analysis revealed distinct profiles over time and between fungal strains (Figure 1).

By performing proteomic analyses of the secretomes using LC-MS/MS and mass matching, we detected a high number of secreted proteins (between 150 and over 400 different proteins, depending on the culture conditions; Supplementary Data Set; Figure 2A) encompassing a large diversity of enzymes. CAZymes accounted for ca. 29-52% of the total protein abundance in each secretome (Figure S3), with a notable high diversity of enzyme from the AA class (Figure 2B). Among them, a variety of laccases (AA1), peroxidases (AA2), flavo-oxidases (AA3) including cellobiose dehydrogenases (AA3_1-AA8), CROs (AA5), oligosaccharide oxidases (AA7), lytic polysaccharide monooxygenases (LPMOs; AA9, AA11, AA13, AA16) and pyrroloquinoline quinone-dependent oxidoreductase (AA12) were detected. To determine whether this high number and diversity of proteins detected was the results of cell lysis (7, 48), we evaluated the proportion of proteins predicted as not secreted – i.e., without signal peptide – and checked more specifically the abundance of some known intracellular proteins (i.e., DNA-polymerase, proteasome endopeptidase complex, citrate synthase, malate dehydrogenase, transketolase and ubiquitin-protein ligase) (Figure S4). Despite the significant number of proteins predicted without signal peptide, we did not observe any clear trend that would account for an accumulation of intracellular proteins in the secretomes over time. Notably, some proteins without signal peptide are known to be secreted through unconventional secretion pathways in fungi (48, 49).
162

Effect of Copper-Manganese-Ethanol supplementation on the secretome profiles

We then looked at the effect of supplementation with CME. At first glance, no clear effect was distinguishable on CAZymes diversity (Figures 2 & S3) but when we focused on copper-containing enzymes (Figure 3), a clear trend became apparent. Indeed, copper-containing enzymes were generally more abundant in the CME medium with a diversity of oxidases from the AA class including AA1 laccases, AA5_1 GLOX and AA9, AA11, AA13 and AA16 LPMOs. Furthermore, AA1_3, AA5_1 and AA9 were among the top-five most abundant enzymes in YG-CME for the *C. tabacum* and *C. destructivum* secretomes, while no AA enzymes were identified in the top-five ranking for the corresponding YG secretomes (Table S1). Remarkably, the presence of some AA9-CBM18 was detected in both the *C. tabacum* and *C. destructivum* secretomes (Figure 3). This association of a chitin-binding domain from the CBM18 family (mainly found associated to chitin-active enzymes) to an AA9 domain is intriguing since AA9 LPMOs have not yet been described to act on chitin substrates. Strikingly AA9 LPMOs were particularly abundant at day 3 in YG medium for *C. destructivum* while drastically decreasing at day 7 and 14 (Figure 3) as already observed in the secretomes of the basidiomycete fungus *Laetisaria arvalis* (50). Interestingly, *C. tabacum* AA9 LPMOs were also found in great abundance at all time points in the YG-CME conditions.

In addition, a diversity of Glycoside Hydrolases (GH), Polysaccharide Lyases (PL), Carbohydrate Esterase (CE) and AAs were detected exclusively in the CME medium (Figure 4). Among these enzymes, we noticed the intriguing presence of hydrolases (GH18, GH18-CBM18), oxidases (AA7, AA11) and esterases (CE4-CBM18) potentially active on chitin or chito-oligosaccharides. Remarkably, a multimodular protein with five consecutive CBM50
185 chitin-binding domains (“CBM50 (x5)” in Figure 4) but without any catalytic domain was
186 detected in both the *C. tabacum* and *C. destructivum* secretomes. This protein is analogous to
187 other chitin-binding proteins (i.e., LysM effector proteins) (see discussion).

188 Presence of AA5 in the secretomes

189 We next focused our analysis on AA5 CROs. Interestingly, a putative GLOX from the
190 AA5_1 subfamily was detected in *C. destructivum* secretomes (Figure 5), being the most
191 abundant protein in the YG-CME secretome at day 7 (and the second most abundant at day 14;
192 Table S1). The *C. tabacum* AA5_1 putative GLOX was also the third most abundant protein in
193 the YG-CME secretome at day 14 (Table S1). We could also detect the occurrence of putative
194 AlcOx and AAO from the AA5_2 subfamily in both *C. graminicola* and *C. tabacum* secretomes
195 (Figure 5). However, their relative abundance was quite low (between 0.05-0.13% of the total
196 Protein Abundance Index (PAI)) and they did not accumulate over time. No correlation with the
197 presence of CME in the medium was seen except for the *C. graminicola* AAO. CROs are known
198 to be activated by peroxidases (41, 51, 52). More specifically, the peroxidase partner responsible
199 for the activation of CRO-AlcOx has been recently unveiled in fungal pathogens (20). Both
200 genes occur as a tandem in the genomes of the *Colletotrichum* and *Magnaporthe* species
201 harboring an AlcOx, and are co-expressed *in vivo* prior to the plant-penetration stage (20). While
202 some peroxidases from the AA2 family (see Figure 2) were found in some of the secretomes, the
203 specific peroxidase partner of the AlcOx was not detected reinforcing the authors claims that
204 interaction with plant is needed for genes co-expression (20).

205 Together, these data provide unexpected information on the induction of secreted copper-
206 enzymes in *Colletotrichum* species. However, the low level (or even the absence for *C.
207 destructivum*) of AA5_2 detected under these experimental conditions indicates that the direct
208 implementation of such secretomes for the bioconversion of fatty alcohols is not a viable
209 strategy. This was confirmed by preliminary conversion assays we carried out using the
210 secretomes directly on a range of primary alcohols (data not shown). These tests were
211 inconclusive, most likely due to the low level of AA5_2 secreted and were difficult to interpret
212 given the complexity of the secretome samples (hundreds of enzymes, including various
213 oxidases such as AA3 enzymes – Figure 2B). These results prompted us to produce the
214 *Colletotrichum* CRO-AlcOx enzymes recombinantly in *Pichia pastoris* and to test their ability to
215 convert alcohols of interest for the flavor and fragrance industries. The objective was to find
216 CRO-AlcOx with potential interesting traits not predictable by sequence analysis such as (i)
217 activity on unactivated fatty alcohols, (ii) minimized requirement in accessory enzymes or (iii)
218 reduced overoxidative activity.

219 **Recombinant production and substrate specificity of CdeAlcOx and CtaAlcOx**

220 Detailed examination of the *CdeAlcOx* and *CtaAlcOx* sequences showed that the two
221 AlcOx amino acid sequences share 90% identity with the *CgrAlcOx* and likewise comprise a
222 single catalytic module, as with all other characterized CRO-AlcOx from *Colletotrichum* spp.
223 (Figure S1). The two AAO share 85% identity with the recently characterized *CgrAAO* (44) and
224 possess a N-terminal PAN_1 domain, in addition to the catalytic module, similarly to the PAN_1
225 domain observed in the *CgrAAO* (Figure S1). The function of the PAN_1 domain in AA5_2-
226 AAOs is unclear but such modules are known to mediate protein-protein or protein-carbohydrate
227 interactions (53). We successfully produced both CRO-AlcOx in *P. pastoris* (Figure S5) with
228 high titers for *CtaAlcOx* in flask culture (200 mg of purified enzyme per liter of culture)
229 compared to *CdeAlcOx* (75 mg.L⁻¹) and *CgrAlcOx* (35 mg.L⁻¹). Activity screening using the
230 ABTS/HRP assay, with benzyl alcohol (BnOH - often use as benchmark with CRO-AlcOx (18,
21, 44)) used as reference, confirmed that both enzymes are indeed AlcOx and that they show
232 similar substrate specificities compared to the canonical CgrAlcOx (Figure 6). However,
233 CdeAlcOx displayed a slightly higher specific activity on the secondary alcohol butan-2-ol, and
234 on the longer aliphatic substrate decan-1-ol. This latter observation could be related to the
235 M173L substitution compared to CgrAlcOx, that could create a slightly more hydrophobic
236 environment in the active site, favoring the accommodation of bulky alcohol substrates (Figure
237 S6). The pH-rate profiles determined for CdeAlcOx and CtaAlcOx (Figure S7), revealed pH
238 optima around 7.5 – 8 for both enzymes. For subsequent conversion assays and comparison with
239 CgrAlcOx, only CdeAlcOx was retained because CtaAlcOx exhibited no noticeable differences
240 in terms of substrate specificity. Nevertheless, CtaAlcOx remains attractive from a
241 biotechnological point of view, being 5.7-fold more highly expressed than the CgrAlcOx in flask
242 cultures.

243 Biocatalytic production of odorant aldehydes using CdeAlcOx

244 CdeAlcOx was subsequently probed for the conversion of hexan-1-ol and octan-1-ol (two
245 precursors of fragrant aldehydes) using CgrAlcOx as a benchmark. For these experiments, we
246 tested the impact of the reaction time (15 minutes versus 16 hours conversion) and the addition
247 of accessory enzymes (HRP and catalase) according to the parameters determined in a previous
248 study (21). Conversion results showed that CdeAlcOx behave similarly to CgrAlcOx with all the
249 tested substrates (Figure 7). As previously observed with CgrAlcOx (21, 33), CdeAlcOx alone
250 was unable to promote full alcohol conversion. However, when both HRP and catalase were
251 added to the reaction mixture, full conversion was readily achieved in 15 minutes. Strikingly, the
252 sole addition of catalase to the reaction mixture resulted in a significant improvement in substrate
253 conversion that probably cannot be attributed only to its protective effect against H₂O₂. This may
254 suggest an activating effect of catalase on CRO-AlcOx, similar to that of HRP.

255 We have previously demonstrated that upon long reaction times (16 hours), partial
256 overoxidation of fatty aldehydes >C6 was observed due to HRP, most likely through a non-
257 enzymatic mechanism (Figure 7A) (21). Carboxylic acids are undesired products in the frame of
258 the F&F application targeted here, but are valuable molecules in other fields (54). In agreement
259 with our previous study (21), we observed here that carboxylic acid was not noticeable with
260 hexan-1-ol as substrate (Figure 7B), but was observed after 16 hours of conversion of octan-1-ol,
261 only when HRP was present in the reaction (Figure 7C).

262 We also observed previously (21) that in the case of benzaldehyde derivatives (i.e. 4-
263 NO₂-benzyl alcohol) some overoxidation was detected but mediated by the CgrAlcOx itself
264 (Scheme S1) and dependent according to the propension of the aldehyde product to undergo
265 hydration. To probe whether CdeAlcOx behave in the same way as CgrAlcOx with 4-NO₂-
266 benzyl alcohol, we included this substrate in the panel of compounds assayed (Figure S8). The
267 conversion of 4-NO₂-benzyl alcohol indeed led to the corresponding acid after 16 hours of
268 reaction even when HRP is omitted from the reaction (Figure S8). Altogether, these results
269 confirm that the behavior observed for the CgrAlcOx regarding accessory enzymes and
270 overoxidation (21) applies to other CRO-AlcOx enzymes.

271

272

273 DISCUSSION

274 The time-resolved proteomic analyses performed here on *Colletotrichum* secretomes
275 revealed interesting and unexpected results. We detected up to ca. 400 individual enzymes in the
276 secretomes, with the maximum number found in the YG-CME medium, while former studies by
277 our group usually yielded ca. 80 – 250 secreted proteins (8, 10, 55, 56). The high proportion of
278 CAZymes in the secretomes is surprising considering the absence of any inducer to mimic plant
279 cell walls. Strikingly, the addition of CME to the medium seemed to boost the secretion of AA
280 enzymes. Moreover, our finding that some LPMOs (especially AA9) preferentially accumulated
281 in the CME medium, in the absence of any plant biomass raises questions about the molecular
282 basis of the regulatory pathways driving their production, and possibly their true biological
283 function (57). In line with this observation, recent work suggests that some fungal LPMOs and
284 LPMO-like proteins may be involved in biological functions distinct from plant biomass
285 conversion, i.e., copper homeostasis and pathogenicity (58, 59) as well as fungal cell fusion (60).
286 Other copper-containing AA enzymes exhibited higher accumulation in YG-CME cultures.
287 Induction of the secretion of laccases could either be due to the addition of ethanol to the
288 medium (12), or a response to oxidative stress (61). Indeed, it is known that at high
289 concentrations, copper contributes to generate reactive oxygen species by Fenton chemistry (62,
290 63).

291 The proteins secreted exclusively in YG-CME medium revealed a range of chitin-active
292 enzymes, as well as non-enzymatic chitin-binding proteins (LysM proteins) some of which are
293 released by fungal cells upon interaction with their plant hosts, to conceal their presence from the
294 host immune system by preventing fungal chitin recognition by the plant or protecting fungal
295 hyphae against plant chitinases (64–66). In the absence of the host or of any plant biomass, this
296 observation suggests that the high concentration of CME in the medium could trigger some
297 pathways related to pathogenesis. The presence of chitin active enzymes may also be indicative
298 of a nutrient recycling in older senescing portions of the mycelium or of some fungal cell wall
299 remodeling activity (67).
The relatively high abundance of GLOX from the AA5_1 subfamily in some YG-CME secretomes and the low secretion levels of AA5_2 AlcOx suggest distinct biological functions for these two types of CRO. AA5_1 GLOX are often described as enzymes acting on lignin in cooperation with some peroxidases in wood-decaying fungi (68). However, their high abundance here, under CME conditions, despite the absence of lignin points toward a possible additional biological function of GLOX in Ascomycota, related to filamentous growth or pathogenicity, as already demonstrated for the AA5_1 GLOX from the maize pathogen *Ustilago maydis* (69).

The primary objective of this study was to investigate the secretion of CRO-AlcOx by natural fungal strains with bioconversion potential. Under our experimental conditions, CRO-AlcOx are only sporadically secreted. Of note, a separate study very recently investigated the biological function of these enzymes and showed that they are co-secreted at low levels, over a short period of time during penetration of the plant host cuticle, and spatially restricted to the puncturing site (20). Thus, with hindsight, the secretion of large amount of CRO-AlcOx might be difficult to achieve without a proper signal to mimic the presence of plant host. Another option would be to identify and engineer the promoter region controlling co-expression of the genes encoding both the AlcOx and the associated peroxidase. However, for biocatalytic purposes, the recombinant expression of CRO-AlcOx remains the most promising alternative. We here confirmed that the knowledge gathered on sequence-structure-function of AA5_2 (18, 21, 44), can predict an AlcOx activity based on bioinformatic analysis and that various CRO-AlcOx can be easily produced heterologously using the yeast *P. pastoris* as host. *CtaAlcOx* was produced to high yield, which is promising for future upscaled production. The conversion assays performed with *CdeAlcOx* revealed that this enzyme can be used as a biocatalyst for the conversion of fatty alcohols. The behavior of *CdeAlcOx* in terms of overoxidation and accessory enzyme
323 requirements suggests that the traits observed here and previously (20, 21, 33) are probably valid
324 for all CRO-AlcOx.

325 In conclusion, the work carried out in this study highlights that *Colletotrichum* species
326 can secrete a range of copper-containing oxidoreductases from the AA class without any plant
327 cell wall inducer. While CRO-AlcOx were detected in some of the secretomes, the direct
328 utilization of fungal strains for bioconversion of fatty alcohols will require further efforts to
329 achieve a high secretion yield of CRO-AlcOx and a deeper understanding of the factors
330 controlling their secretion. The recombinant pathway therefore represents the most promising
331 approach to develop their use in biocatalytic applications for the production of fragrant fatty
332 aldehydes.

333
334 MATERIAL AND METHODS

335 General information

336 All chemicals used for cultivation of fungi and microorganisms were reagent grade or
337 higher. All chemicals used for biochemical assays were ≥ 95% purity. HRP type II and catalase
338 from bovine liver were purchased from Sigma-Aldrich (Hamburg, Germany). Molar
339 concentrations of HRP (MW: 33.89 kDa) and catalase (MW of monomer: 62.5 kDa) were
340 estimated by Bradford assay using BSA as standard. Pre-cast 10% polyacrylamide gel Mini-
341 PROTEAN TGX were purchased from Bio-Rad (USA).

342 Data availability

343 The protein sequences of the CdeAlcOx and CtaAlcOx were deposited in GenBank under
344 the accession numbers MZ269520 and MZ269521 respectively.

345 Natural and recombinant strains

346 The following fungal strains were used: C. graminicola (M1.001, CBS 130836, (35)), C.
347 destructivum (LARS 709, CBS 520.97, (70)) and C. tabacum (N150, CPC 18945, (70)). The
348 DNA sequences of CdeAlcOx and CtaAlcOx were obtained by blasting against the “raw”
349 unannotated genomes of C. destructivum and C. tabacum and further intron prediction was done
350 using Augustus web tool (http://bioinf.uni-greifswald.de/augustus/). DNA cloning and
351 transformation of P. pastoris X33 with codon-optimized genes (for expression in P. pastoris)
352 encoding CdeAlcOx and CtaAlcOx was carried out as described previously for other
353 constructions (71). The recombinant Pichia pastoris X33 strains containing CgrAlcOx gene
354 (GenBank EFQ30446.1) was recovered from previous work (18, 21). The selection of the most
355 productive recombinant transformants was done through culture and expression in 24-deepwell
356 plates with subsequent purification from supernatant by affinity chromatography on Ni-NTA
357 resin using an in-house automated procedure (71).

358
359 Fungal cultures and secretomes harvesting

360 Fungal strains were grown and let sporulated on Potato Dextrose Agar medium (PDA)
361 plates in closed Petri dishes, at 23°C for seven days under natural solar light, without controlling
362 the humidity and were stored at 4°C before utilization. Inocula were prepared by aseptically
363 homogenizing the content of a 90 mm petri dish cultures of *Colletotrichum* (containing fungal
364 biomass (both mycelia and spores) and PDA medium) using an Ultra Turrax T25 homogenizer
365 (IKA, Germany) for 1 min at 9,500 rpm in 80 mL of the culture medium (see below). 200 µL of
366 this homogenate was used to inoculate 100 mL of liquid medium placed in 500 mL baffled flask.

367 Liquid cultures were grown in two different media, namely: "YG" and "YG-CME". YG
368 medium was composed of glucose (10 g.L⁻¹), ammonium tartrate (2 g.L⁻¹), yeast extract (1 g.L⁻¹),
369 K₂HPO₄ (1 g.L⁻¹), MgSO₄·7H₂O (0.5 g.L⁻¹), KCl (0.5 g.L⁻¹) and 0.5 mL.L⁻¹ of trace elements
370 stock solution composed of (NH₄)₆Mo₇O₂₄·4H₂O (20 mg.mL⁻¹), FeSO₄·7H₂O (100 mg.mL⁻¹),
371 ZnSO₄·7H₂O (140 mg.mL⁻¹), B₄O₇Na₂·10H₂O (200 mg.mL⁻¹). Glucose was autoclaved
372 separately as a 10X stock solution. The trace elements stock solution was sterilized by filtration
373 through a 0.22 µm polyether sulfone membrane (Merck-Millipore, Germany). Note that
374 insoluble elements present in this solution were removed by the filtration step. YG-CME
375 medium was prepared exactly as YG but was further supplemented with CuSO₄·5H₂O (500 µM),
376 MnCl₂ (500 µM) and 9 g.L⁻¹ ethanol, filtered sterilized (except for ethanol).

377 The inoculated cultures were placed in an Innova 42R incubator (New Brunswick, USA)
378 and let grown at 25°C under natural light and shaking (at 150 rpm for C. destructivum and 105
379 rpm for *C. tabacum* and *C. graminicola*). The secretomes were harvested at day 3, day 7 and day
380 14 after inoculation. All samples were prepared in triplicates which were pooled upon
381 harvesting. Each harvested secretome was submitted to several filtration steps: (i) Miracloth (22-
382 25 µm pore size) (Merck-Millipore), (ii) several glass microfiber filters used sequentially: 47
383 mm Whatman® grade GF/D, A and F (GE Healthcare, USA), (iii) 0.45 and 0.22-µm polyether
384 sulfone membranes (Merck -Millipore) and finally diafiltered onto a polyether sulfone
385 membrane with a 10kDa cut-off (Vivaspin, Sartorius, Göttingen, Germany) with 50 mM sodium
386 phosphate buffer pH 7.0 to a final volume of 1-7 mL. The secretomes were stored at 4°C until
387 use. Their total protein concentration was evaluated by the Bradford method (Protein Assay,
388 BioRad, France) using a bovine serum albumin (BSA) standard protein.

389

390 Microfluidic capillary gel electrophoresis

391 The concentrated secretomes samples were diluted so that 87.5 µL of the initially
392 harvested secretomes (before concentration) were submitted to electrophoresis, except for *C.*
393 *tabacum* YG-CME day 14 which was further diluted by 1/3 to avoid too high protein
394 concentration. Samples were prepared in a 96-well plate using the manufacturer “HT Protein
395 Express Assay Sample Buffer” (PerkinElmer, USA) supplemented with 36.4 mM (final
396 concentration) of dithiothreitol. The samples were heated at 95°C for 10 min and subjected
397 directly to LabChip GX II capillary gel electrophoresis (PerkinElmer) according to the
398 manufacturer’s instructions. The electropherograms were analyzed using the LabChip GX II
399 software (PerkinElmer).

400

401 LC-MS/MS protein identification
For each secretome, 15 µg of proteins were loaded on a 10% Tris–glycine precast SDS-PAGE gel (Mini-PRO-TEAN TGX, BioRad). After a short migration (0.5 cm) in the stacking gel, the gels were stained with Coomassie blue (BioRad) and each electrophoresis track was cut into two 2-mm-wide strips. Proteomic identification was performed at the Plateforme d'Analyse Protéomique de Paris Sud-Ouest (PAPPSO, INRA, Jouy-en-Josas, France; http://pappso.inra.fr/), according to the protocol described in (72). Briefly, the digestion of the proteins contained in the gel strips was carried out according to a standard trypsinolysis process, using modified trypsin (Promega, France). Peptide analysis was performed by a NanoLC Ultra 2D system (Eksigent Technologies, United States) coupled to a Q-exactive mass spectrometer (Thermo Fisher Scientific, France) using electro-spray ionization. Peptide attribution and protein annotation were performed by comparing mass spectrometry data to predicted proteins in the genomes of *Colletotrichum graminicola* M1.001 (https://mycocosm.jgi.doe.gov/Colgr1/Colgr1.home.html) and *Colletotrichum higginsianum* IMI 349063 (https://mycocosm.jgi.doe.gov/Colhig2/Colhig2.home.html) for both *C. destructivum* and *C. tabacum* strains. The internal contaminant database X!TandemPipeline software (X!TandemPipeline version 0.2.30, France) was also used. The protein annotation was completed manually using the CAZy database for the CAZymes annotation and using the BlastP tool from the NCBI database (https://blast.ncbi.nlm.nih.gov) for other proteins.

420

421 **Heterologous expression and purification**

Recombinant *P. pastoris* X33 strains were streaked on yeast extract peptone dextrose (YPD) agar plates containing Zeocin (100 µg.mL⁻¹) and incubated 3 days at 30°C. Five mL of YPD broth, in 50-mL sterile conical tubes, were inoculated with a *P. pastoris* transformant and
incubated for five hours (30°C, 160 rpm in an orbital shaker). The preculture was used to inoculate 0.2% (v/v) of 500 mL buffered complex glycerol medium (BMGY) in a 2-liter flask, incubated (16 hours) until the OD600 nm reached 4-6. Then, the biomass was harvested by centrifugation (10 minutes, 16°C, 5,000 x g), resuspended in 100 mL of buffered complex methanol medium (BMMY) supplemented with CuSO₄ (500 µM) and methanol (1% v/v) in a 500 mL flask, and shaken for 3 days in an orbital shaker (200 rpm, 16°C) with daily feeding of methanol (1% v/v). The culture was then centrifuged (10 minutes, 4°C, 5,000 x g) and the supernatant containing the secreted proteins, filtrated on a 0.45-µm cut-off membrane (Millipore, USA) followed by buffer exchange with Tris-HCl (50 mM, pH 8.5), performed by ultrafiltration through a 10 kDa cut-off polyether sulfone membrane (Vivacell 250, Sartorius Stedim Biotech GmbH, Germany). The buffered supernatant was loaded on a DEAE-20 mL HiPrep FF 16/10 anion exchange chromatography column (GE Healthcare), equilibrated with Tris-HCl buffer (50 mM, pH 8.5) and connected to an Äkta Xpress system (GE Healthcare). Elution was performed by applying a linear gradient from 0 to 500 mM NaCl (in Tris-HCl buffer 50 mM, pH 8.5) with a flow rate set to 5 mL.min⁻¹. The collected fractions were analyzed by SDS-PAGE (10% polyacrylamide precast gel, Bio-Rad) stained with Coomassie blue. Fractions containing the recombinant enzyme were pooled, concentrated and exchanged to sodium phosphate buffer (50 mM, pH 7.0). The CgrAlcOx (ε²⁸⁰ = 101,215 M⁻¹.cm⁻¹), CdeAlcOx (ε²⁸⁰ = 94,225 M⁻¹.cm⁻¹) and CtaAlcOx (ε²⁸⁰ = 94,225 M⁻¹.cm⁻¹) concentration was determined by UV absorption at 280 nm using a Nanodrop ND-200 spectrophotometer (Thermo Fisher Scientific). Final solutions (3-5.5 mg.mL⁻¹) were flash-frozen using liquid nitrogen and stored at -80°C for long-term storage or stored at 4°C for immediate use.
448 **Spectrophotometric enzyme activity assays**

449 AlcOx initial rates were determined by monitoring H₂O₂ released during AlcOx oxidation
450 of various substrates, using the coupled ABTS/HRP assay. Routine assays were performed in 96-
451 wells transparent microtiter plates (Flat bottom, Polystyrene - Greiner Bio One, Austria) in 200
452 µL final volume containing 0,25 mg/mL ABTS powder, 0,1 mg.mL⁻¹ HRP powder (as provided
453 by the supplier), 3 mM substrate (unless indicated otherwise) and 1 nM AlcOx in sodium
454 phosphate buffer (50 mM, pH 8.0), at 23°C. Reactions were initiated by the addition of substrate
455 to a premix containing all other reagents. Evolution of the absorbance at 414 nm (ABTS cation
456 radical) was measured over time with a Tecan Infinite M200 (Tecan, Switzerland) plate reader.
457 Oxidation of 1 mole of substrate by AlcOx consumes 1 equivalent of O₂ and generates 1
458 equivalent of H₂O₂ which is in turn used by HRP as co-substrate to oxidize 2 equivalents of
459 ABTS. Insoluble substrates were prepared in acetone so that the final acetone concentration in
460 the enzyme assay did not exceed 1% (v/v). Standard curve of known concentrations of H₂O₂ (1 to
461 40 µM) were made and used to quantify H₂O₂ production. One unit of AlcOx activity was
462 defined as the amount of enzyme necessary to produce 1 µmole of H₂O₂ per minute.

463
464 **Bioconversion assays**

465 Bioconversions were carried out in 4 mL-clear borosilicate glass vials closed by screw
466 caps with PTFE septum for a total reaction volume of 500 µL. Routine assay contained 3 mM
467 substrate (prediluted in acetone), 1 µM AlcOx, various amounts of catalase (0-8 µM) and/or
468 HRP (0-12 µM), in sodium phosphate buffer (50 mM, pH 8.0). Final reaction contained 1% (v/v)
469 acetone. Reactions were run at 23°C, under stirring at 190 rpm in an Innova 42R incubator (New
470 Brunswick, USA). Vials were placed lying down in the incubator. The reaction mixture was then
471 acidified by addition of 10 µL HCl (12 M) and products and possible remaining substrate were
472 extracted by adding 500 µL of cyclohexane/ethyl acetate mixture (1:1), followed by shaking and
473 centrifugation for 5 minutes at 1.500 x g. The organic layer was transferred into a new vial by
474 pipetting and analyzed with a GC-2014 apparatus (Shimadzu, Japan) equipped with a flame
475 ionization detector (FID) and an Optima-σ-3 GC capillary column (30 m x 0.25 mm x 0.25 µm -
476 Macherey-Nagel GmbH & Co, Germany). Nitrogen (200 kPa) was used as carrier gas. The
477 injector and detector temperatures were set at 250°C and temperature programs are described in
478 Table S2. Heptanal or guaiacol were used as internal standards for either aliphatic or aromatic
479 products analyses, respectively.

480
481 AUTHOR CONTRIBUTIONS

482 D.R. carried out most of the experimental work, analyzed the data. S.S. was involved in
483 the culture of fungi and production of the secretomes. L.O.C. and D.N. performed proteomics
484 and analysed data. E.D. and B.H. performed expert annotation of CAZymes. M.H. contributed to
485 enzyme production in *Pichia pastoris*. S.G. performed the microfluidic capillary gel
486 electrophoresis experiments. R.J.O. and N.L. provided the *Colletotrichum* strains and access to
487 the associated genomic data. M.L. and J-G.B. conceptualized the study. F.L., B.B., M.L. and J-
488 G.B. supervised the work. D.R. and J-G.B. drafted the manuscript. All authors approved the final
489 version of the manuscript.

490
491 ACKNOWLEDGEMENTS

492 This study was supported by the French National Agency for Research (“Agence
493 Nationale de la Recherche”) through the “Projet de Recherche Collaboratif International” ANR-
494 NSERC (FUNTASTIC project, ANR-17-CE07-0047). We are grateful to MANE & Fils and the
495 "Association Nationale Recherche Technologie" (ANRT) for funding the PhD fellowship of
496 D.R. entitled "Discovery and structure-function study of new fungal copper radicals oxidases
497 used as biocatalysts for the valorisation of alcohols and plant biomass". This *Convention
498 Industrielle de Formation par la Recherche* (CIFRE) grant no. 2017/1169 runs from 1 April
499 2018 to 1 April 2021. Fungal genome sequencing was supported by funding to R.J.O. (grant
500 ANR-17-CAPS-0004-01).

501 We are grateful to Renaud Vincentelli (AFMB - CNRS - Marseille, France) for providing access
502 to the microfluidic capillary gel electrophoresis apparatus.

503

504 COMPETING INTEREST

505 The authors declare that they have no competing interests.

506

507 REFERENCES

508 1. Wohlgemuth R, Twardowski T, Aguilar A. 2021. Bioeconomy moving forward step by step
509 – A global journey. New Biotechnol 61:22–28.

510 2. Lokko Y, Heijde M, Schebesta K, Scholtès P, Van Montagu M, Giacca M. 2018.

511 Biotechnology and the bioeconomy—Towards inclusive and sustainable industrial
512 development. New Biotechnol 40:5–10.

513 3. Pellis A, Cantone S, Ebert C, Gardossi L. 2018. Evolving biocatalysis to meet bioeconomy
514 challenges and opportunities. New Biotechnol 40:154–169.

515 4. Meyer V, Basenko EY, Benz JP, Braus GH, Caddick MX, Csukai M, de Vries RP, Endy D,

516 Frisvad JC, Gunde-Cimerman N, Haarmann T, Hadar Y, Hansen K, Johnson RI, Keller NP,
517 Kraševec N, Mortensen UH, Perez R, Ram AFJ, Record E, Ross P, Shapaval V, Steiniger
518 C, van den Brink H, van Munster J, Yarden O, Wösten HAB. 2020. Growing a circular
519 economy with fungal biotechnology: a white paper. Fungal Biol Biotechnol 7:5.

520 5. Hage H, Rosso M-N. 2021. Evolution of Fungal Carbohydrate-Active Enzyme Portfolios
521 and Adaptation to Plant Cell-Wall Polymers. 3. J Fungi 7:185.

522 6. Glass NL, Schmoll M, Cate JHD, Coradetti S. 2013. Plant Cell Wall Deconstruction by
523 Ascomycete Fungi. Annu Rev Microbiol 67:477-498.

524 7. Arntzen MØ, Bengtsson O, Várnai A, Delogu F, Mathiesen G, Eijsink VGH. 2020.
525 Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous
526 fungi. 1. Sci Rep 10:20267.

527 8. Couturier M, Navarro D, Olivé C, Chevret D, Haon M, Favel A, Lesage-Meessen L,
528 Henrissat B, Coutinho PM, Berrin J-G. 2012. Post-genomic analyses of fungal
529 lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen
530 *Ustilago maydis*. BMC Genomics 13:57.

531 9. Grandmontagne D, Navarro D, Neugnot-Roux V, Ladevèze S, Berrin J-G. 2021. The
532 Secretomes of *Aspergillus japonicus* and *Aspergillus terreus* Supplement the Rovabio®
533 Enzyme Cocktail for the Degradation of Soybean Meal for Animal Feed. 4. J Fungi 7:278.

534 10. Filiatrault-Chastel C, Navarro D, Haon M, Grisel S, Herpoël-Gimbert I, Chevret D, Fanuel
535 M, Henrissat B, Heiss-Blanquet S, Margeot A, Berrin J-G. 2019. AA16, a new lytic
536 polysaccharide monooxygenase family identified in fungal secretomes. Biotechnol Biofuels
537 12:55.
538 11. Ravalason H, Jan G, Mollé D, Pasco M, Coutinho PM, Lapierre C, Pollet B, Bertaud F,
539 Petit-Conil M, Grisel S, Sigoillot J-C, Asther M, Herpoël-Gimbert I. 2008. Secretome
540 analysis of *Phanerochaete chrysosporium* strain CIRM-BRFM41 grown on softwood. Appl
541 Microbiol Biotechnol 80:719.

542 12. Lomascolo A, Record E, Herpoel-Gimbert I, Delattre M, Robert JL, Georis J, Dauvrin T,
543 Sigoillot J-C, Asther M. 2003. Overproduction of laccase by a monokaryotic strain of
544 *Pycnoporus cinnabarinus* using ethanol as inducer. J Appl Microbiol 94:618-624.

545 13. Lomascolo A, Cayol J-L, Roche M, Guo L, Robert J-L, Record E, Lesage-Meessen L,
546 Ollivier B, Sigoillot J-C, Asther M. 2002. Molecular clustering of *Pycnoporus* strains from
547 various geographic origins and isolation of monokaryotic strains for laccase
548 hyperproduction. Mycol Res 106:1193-1203.

549 14. Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B. 2013. Expansion of the
550 enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes.
551 Biotechnol Biofuels 6:41.

552 15. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. 2014. The
553 carbohydrate-active enzymes database (CAZy) in 2013. D1. Nucleic Acids Res 42:D490-
554 D495.

555 16. Cooper JAD, Smith W, Bacila M, Medina H. 1959. Galactose Oxidase from *Polyporus*
556 *circinatus*, Fr. 3. J Biol Chem 234:445-448.

557 17. Avigad G, Amaral D, Asensio C, Horecker BL. 1962. The d-Galactose Oxidase of
558 *Polyporus circinatus*. 9. J Biol Chem 237:2736-2743.
559 18. Yin D, Urresti S, Lafond M, Johnston EM, Derikvand F, Ciano L, Berrin J-G, Henrissat B,
560 Walton PH, Davies GJ, Brumer H. 2015. Structure–function characterization reveals new
catalytic diversity in the galactose oxidase and glyoxal oxidase family. 1. Nat Commun
561 6:10197.
562

563 19. Oide S, Tanaka Y, Watanabe A, Inui M. 2019. Carbohydrate-binding property of a cell wall
564 integrity and stress response component (WSC) domain of an alcohol oxidase from the rice
blast pathogen *Pyricularia oryzae*. Enzyme Microb Technol 125:13–20.
565

566 20. Bissaro B, Kodama S, Hage H, Ribeaucourt D, Haon M, Grisel S, Simaan J, Beisson F,
567 Forget S, Brumer H, Rosso M-N, O’Connell R, Lafond M, Kubo Y, Berrin J-G. 2021.
568 Unravelling the role of alcohol copper radical oxidases in fungal plant pathogens. Research
569 square

570 21. Ribeaucourt D, Bissaro B, Guallar V, Yemloul M, Haon M, Grisel S, Alphand V, Brumer
571 H, Lambert F, Berrin J-G, Lafond M. 2021. Comprehensive Insights into the Production of
572 Long Chain Aliphatic Aldehydes Using a Copper-Radical Alcohol Oxidase as Biocatalyst.
573 ACS Sustainable Chem Eng 9:4411–4421.

574 22. Liu J, Wu S, Li Z. 2018. Recent advances in enzymatic oxidation of alcohols. Curr Opin
575 Chem Biol 43:77–86.

576 23. Dong J, Fernández-Fueyo E, Hollmann F, Paul CE, Pesic M, Schmidt S, Wang Y, Younes
577 S, Zhang W. 2018. Biocatalytic Oxidation Reactions: A Chemist’s Perspective. 30. Angew
578 Chem, Int Ed 57:9238–9261.
579 24. Surburg H, Panten J. 2016. Common Fragrance and Flavor Materials: Preparation, 580 Properties and Uses, 6th ed. Wiley-VCH.

581 25. Tsuzuki S. 2019. Higher Straight-Chain Aliphatic Aldehydes: Importance as Odor-Active 582 Volatiles in Human Foods and Issues for Future Research. 17. J Agric Food Chem 583 67:4720-4725.

584 26. Burdock GA. 2016. Fenaroli's Handbook of Flavor Ingredients. CRC Press.

585 27. Rouseff R, Perez-Cacho PR. 2007. Citrus Flavour, p. 117-134. In Berger, RG (ed.), 586 Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Springer, Berlin, 587 Heidelberg.

588 28. Kohlpaintner C, Schulte M, Falbe J, Lappe P, Weber J, Frey GD. 2013. Aldehydes, 589 Aliphatic. Ullmann's Encyclopedia of Industrial Chemistry. American Cancer Society.

590 29. Shah J, Arslan E, Cirucci J, O'Brien J, Moss D. 2016. Comparison of Oleo- vs Petro- 591 Sourcing of Fatty Alcohols via Cradle-to-Gate Life Cycle Assessment. J Surfactants Deterg 592 19:1333-1351.

593 30. Baldwin E, Plotto A, Manthey J, McCollum G, Bai J, Irey M, Cameron R, Luzio G. 2010. 594 Effect of Liberibacter Infection (Huanglongbing Disease) of Citrus on Orange Fruit 595 Physiology and Fruit/Fruit Juice Quality: Chemical and Physical Analyses. 2. J Agric Food 596 Chem 58:1247-1262.
597 31. Dala-Paula BM, Plotto A, Bai J, Manthey JA, Baldwin EA, Ferrarezi RS, Gloria MBA.

598 2019. Effect of Huanglongbing or Greening Disease on Orange Juice Quality, a Review.

599 Front Plant Sci 9.

600 32. Ribeaucourt D, Bissaro B, Lambert F, Lafond M, Berrin J-G. 2021. Biocatalytic oxidation

601 of fatty alcohols into aldehydes for the flavors and fragrances industry. Biotechnol Adv.

602 107787.

603 33. Forget S, Xia R (Fan), Hein JE, Brumer H. 2020. Determination of biocatalytic parameters

604 of a copper radical oxidase using real-time reaction progress monitoring. Org Biomol Chem

605 18:2005-2184.

606 34. Baroncelli R, Amby DB, Zapparata A, Sarrocco S, Vannacci G, Le Floch G, Harrison RJ,

607 Holub E, Sukno SA, Sreenivasaprasad S, Thon MR. 2016. Gene family expansions and

608 contractions are associated with host range in plant pathogens of the genus *Colletotrichum*.

609 1. BMC Genomics 17:555.

610 35. O'Connell RJ, Thon MR, Hacquard S, Amyotte SG, Kleemann J, Torres MF, Damm U,

611 Buiate EA, Epstein L, Alkan N, Altmüller J, Alvarado-Balderrama L, Bauser CA, Becker

612 C, Birren BW, Chen Z, Choi J, Crouch JA, Duvick JP, Farman MA, Gan P, Heiman D,

613 Henrissat B, Howard RJ, Kabbage M, Koch C, Kracher B, Kubo Y, Law AD, Lebrun M-H,

614 Lee Y-H, Miyara I, Moore N, Neumann U, Nordström K, Panaccione DG, Panstruga R,

615 Place M, Proctor RH, Prusky D, Rech G, Reinhardt R, Rollins JA, Rounsley S, Schardl CL,

616 Schwartz DC, Shenoy N, Shirasu K, Sikhakolli UR, Stüber K, Sukno SA, Sweigard JA,

617 Takano Y, Takahara H, Trail F, van der Does HC, Voll LM, Will I, Young S, Zeng Q,

618 Zhang J, Zhou S, Dickman MB, Schulze-Lefert P, Ver Loren van Themaat E, Ma L-J,
619 Vaillancourt LJ. 2012. Lifestyle transitions in plant pathogenic *Colletotrichum* fungi 620 deciphered by genome and transcriptome analyses. 9. Nat Genet 44:1060–1065.

621 36. Perfect SE, Hughes HB, O'Connell RJ, Green JR. 1999. *Colletotrichum*: A Model Genus 622 for Studies on Pathology and Fungal-Plant Interactions. Fungal Genet Biol 27:186–198.

623 37. Armesto C, Maia FGM, Monteiro FP, Abreu MS de. 2020. exoenzymes as a pathogenicity 624 factor for *Colletotrichum gloeosporioides* associated with coffee plants. Summa 625 phytopathol 45:368–373.

626 38. Onofre SB, Steilmann P, Bertolini J, Rotta D, Francini AS, Kagimura Y, Acirc S, Groff N, 627 Mazzali L. 2011. Amylolytic enzymes produced by the fungus *Colletotrichum* 628 gloeosporioides in rice semi-solid fermentation. J Yeast Fungal Res 2:28–32.

629 39. Tokuyasu K, Ohnishi-Kameyama M, Hayashi K. 1996. Purification and Characterization of 630 Extracellular Chitin Deacetylase from *Colletotrichum lindemuthianum*. Biosci, Biotechnol, 631 Biochem 60:1598–1603.

632 40. Rispoli FJ, Shah V. 2007. Mixture design as a first step for optimization of fermentation 633 medium for cutinase production from *Colletotrichum lindemuthianum*. J Ind Microbiol 634 Biotechnol 34:349–355.

635 41. T. Pedersen A, Birmingham WR, Rehn G, Charnock SJ, Turner NJ, Woodley JM. 2015. 636 Process Requirements of Galactose Oxidase Catalyzed Oxidation of Alcohols. 11. Org 637 Process Res Dev 19:1580–1589.
638 42. Kersten PJ. 1990. Glyoxal oxidase of *Phanerochaete chrysosporium*: its characterization
639 and activation by lignin peroxidase. 8. PNAS 87:2936–2940.

640 43. Andberg M, Mollerup F, Parikka K, Koutaniemi S, Boer H, Juvonen M, Master E,
641 Tenkanen M, Kruus K. 2017. A Novel *Colletotrichum graminicola* Raffinose Oxidase in
642 the AA5 Family. 20. Appl Environ Microbiol 83.

643 44. Mathieu Y, Offen WA, Forget SM, Ciano L, Viborg AH, Blagova E, Henrissat B, Walton
644 PH, Davies GJ, Brumer H. 2020. Discovery of a fungal copper radical oxidase with high
645 catalytic efficiency towards 5-hydroxymethylfurfural and benzyl alcohols for
646 bioprocessing. ACS Catal 10:3042–3058.

647 45. Copete LS, Chanagá X, Barriuso J, López-Lucendo MF, Martínez MJ, Camarero S. 2015.
648 Identification and characterization of laccase-type multicopper oxidases involved in dye-
649 decolorization by the fungus *Leptosphaerulina sp*. BMC Biotechnol 15.

650 46. Zeiner CA, Purvine SO, Zink EM, Paša-Tolić L, Chaput DL, Haridas S, Wu S, LaButti K,
651 Grigoriev IV, Henrissat B, Santelli CM, Hansel CM. 2016. Comparative Analysis of
652 Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi. 7. PLOS ONE
653 11:e0157844.

654 47. Markus Z, Miller G, Avigad G. 1965. Effect of Culture Conditions on the Production of d-
655 Galactose Oxidase by *Dactylium dendroides*. Appl Microbiol 13:686–693.

656 48. Miura N, Ueda M. 2018. Evaluation of Unconventional Protein Secretion by
657 *Saccharomyces cerevisiae* and other Fungi. Cells 7.
658 49. Vivek-Ananth RP, Mohanraj K, Vandanashree M, Jhingran A, Craig JP, Samal A. 2018.

659 Comparative systems analysis of the secretome of the opportunistic pathogen *Aspergillus*

660 *fumigatus* and other *Aspergillus* species. 1. Sci Rep 8:6617.

661 50. Navarro D, Rosso M-N, Haon M, Olivé C, Bonnin E, Lesage-Meessen L, Chevret D,

662 Coutinho PM, Henrissat B, Berrin J-G. 2014. Fast solubilization of recalcitrant cellulosic

663 biomass by the basidiomycete fungus *Laetisaria arvalis* involves successive secretion of

664 oxidative and hydrolytic enzymes. Biotechnol Biofuels 7:143.

665 51. Cleveland L, Coffman RE, Coon P, Davis L. 1975. Role of the copper in galactose oxidase.

666 6. Biochemistry 14:1108–1115.

667 52. Wohlschlager L, Kracher D, Scheiblbrandner S, Csarman F, Ludwig R. 2021.

668 Spectroelectrochemical investigation of the glyoxal oxidase activation mechanism.

669 Bioelectrochemistry 107845.

670 53. Tordai H, Bányai L, Patthy L. 1999. The PAN module: the N-terminal domains of

671 plasminogen and hepatocyte growth factor are homologous with the apple domains of the

672 prekallikrein family and with a novel domain found in numerous nematode proteins. FEBS

673 Letters 461:63–67.

674 54. Balaraman E, Khaskin E, Leitus G, Milstein D. 2013. Catalytic transformation of alcohols

675 to carboxylic acid salts and H₂ using water as the oxygen atom source. Nature Chem 5:122–

676 125.

677 55. Poidevin L, Berrin J-G, Bennati-Granier C, Levasseur A, Herpoël-Gimbert I, Chevret D,

678 Coutinho PM, Henrissat B, Heiss-Blanquet S, Record E. 2014. Comparative analyses of
679 *Podospora anserina* secretomes reveal a large array of lignocellulose-active enzymes. Appl
680 Microbiol Biotechnol 98:7457–7469.

681 56. Ravalason H, Grisel S, Chevret D, Favel A, Berrin J-G, Sigoillot J-C, Herpoël-Gimbert I.
682 2012. *Fusarium verticillioides* secretome as a source of auxiliary enzymes to enhance
683 saccharification of wheat straw. Bioresour Technol. 114:589–596.

684 57. Agger JW, Isaksen T, Várnai A, Vidal-Melgosa S, Willats WGT, Ludwig R, Horn SJ,
685 Eijsink VGH, Westereng B. 2014. Discovery of LPMO activity on hemicelluloses shows
686 the importance of oxidative processes in plant cell wall degradation. PNAS 111:6287–6292.

687 58. Labourel A, Frandsen KEH, Zhang F, Brouilly N, Grisel S, Haon M, Ciano L, Ropartz D,
688 Fanuel M, Martin F, Navarro D, Rosso M-N, Tandrup T, Bissaro B, Johansen KS, Zerva A,
689 Walton PH, Henrissat B, Leggio LL, Berrin J-G. 2020. A fungal family of lytic
690 polysaccharide monooxygenase-like copper proteins. 3. Nat Chem Biol 16:345–350.

691 59. Garcia-Santamarina S, Probst C, Festa RA, Ding C, Smith AD, Conklin SE, Brander S,
692 Kinch LN, Grishin NV, Franz KJ, Riggs-Gelasco P, Lo Leggio L, Johansen KS, Thiele DJ.
693 2020. A lytic polysaccharide monooxygenase-like protein functions in fungal copper import
694 and meningitis. 3. Nat Chem Biol 16:337–344.

695 60. Gonçalves AP, Heller J, Span EA, Rosenfield G, Do HP, Palma-Guerrero J, Requena N,
696 Marletta MA, Glass NL. 2019. Allorecognition upon Fungal Cell-Cell Contact Determines
697 Social Cooperation and Impacts the Acquisition of Multicellularity. Curr Biol 29:3006-
698 3017.e3.
699 61. Kaur K, Sharma A, Capalash N, Sharma P. 2019. Multicopper oxidases: Biocatalysts in microbial pathogenesis and stress management. *Microbiol Res* 222:1–13.

701 62. Gaetke L. 2003. Copper toxicity, oxidative stress, and antioxidant nutrients. *Toxicology* 189:147–163.

703 63. Kohen R, Nyska A. 2002. Invited Review: Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification. *Toxicol Pathol* 30:620–650.

706 64. Sánchez-Vallet A, Tian H, Rodriguez-Moreno L, Valkenburg D-J, Saleem-Batcha R, Wawra S, Kombrink A, Verhage L, Jonge R de, Esse HP van, Zuccaro A, Croll D, Mesters JR, Thomma BPHJ. 2020. A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization. *PLoS Pathog* 16:e1008652.

710 65. Akcapinar GB, Kappel L, Sezerman OU, Seidl-Seiboth V. 2015. Molecular diversity of LysM carbohydrate-binding motifs in fungi. *Curr Genet* 61:103–113.

712 66. Takahara H, Hacquard S, Kombrink A, Hughes HB, Halder V, Robin GP, Hiruma K, Neumann U, Shinya T, Kombrink E, Shibuya N, Thomma BPHJ, O'Connell RJ. 2016. *Colletotrichum higginsianum* extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity. *New Phytologist* 211:1323–1337.

717 67. Emri T, Molnár Z, Szilágyi M, Pócsi I. 2008. Regulation of autolysis in *Aspergillus nidulans*. *Appl Biochem Biotechnol* 151:211–220.
719 68. Daou M, Faulds CB. 2017. Glyoxal oxidases: their nature and properties. 5. World J
Microbiol Biotechnol 33:87.
720

721 69. Leuthner B, Aichinger C, Oehmen E, Koopmann E, Müller O, Müller P, Kahmann R,
722 Bölker M, Schreier PH. 2005. A H₂O₂-producing glyoxal oxidase is required for
723 filamentous growth and pathogenicity in *Ustilago maydis*. 6. Mol Genet Genomics
724 272:639-650.

725 70. Damm U, O'Connell RJ, Groenewald JZ, Crous PW. 2014. The *Colletotrichum
destructivum* species complex – hemibiotrophic pathogens of forage and field crops. Stud
726 Mycol 79:49–84.
727

728 71. Haon M, Grisel S, Navarro D, Gruet A, Berrin J-G, Bignon C. 2015. Recombinant protein
729 production facility for fungal biomass-degrading enzymes using the yeast *Pichia pastoris*.
730 Front Microbiol 6.

731 72. Amara S, Perrot T, Navarro D, Deroy A, Benkhelfallah A, Chalak A, Daou M, Chevret D,
732 Faulds CB, Berrin J-G, Morel-Rouhier M, Gelhaye E, Record E. 2018. Enzyme Activities
733 of Two Recombinant Heme-Containing Peroxidases, *Tv* DyP1 and *Tv* VP2, Identified from
734 the Secretome of *Trametes versicolor*. Appl Environ Microbiol 84:e02826-17,
735 /aem/84/8/e02826-17.atom.

736
737 FIGURE LEGENDS
738

738
729 **Figure 1.** Analysis by capillary electrophoresis of the *Colletotrichum secretomes* samples.
730 Concentrated secretomes samples were diluted so that the equivalent of 87.5 µL (prior to
731 concentration) of each secretome sample was injected, except for *C. tabacum* YG-CME day 14
732 that was further diluted 3-fold to avoid a too high protein concentration. The molecular weights
733 (in kDa) of the protein ladder are displayed on the left- and right-hand sides of the image. A darker
734 color corresponds to a higher concentration of protein.

735
736 **Figure 2: Overview of (A) the total number of proteins secreted over time in different media and (B) diversity of AA enzymes detected in the secretomes.**
737
738

739 **Figure 3: Abundance of copper-containing AAs in fungal secretomes from (A) C. graminicola, (B) C. destructivum and (C) C. tabacum.** The Y-axis legend on panel A applies to all panels likewise (note the different scale for C. tabacum). For each enzyme family, the bar is subdivided according to the number of enzymes identified; for instance, C. tabacum at day 3 in YG-CME displayed six different AA9. PAI: Protein Abundance Index.

744 **Figure 4.** Abundance of CAZymes identified exclusively in the YG-CME media from (A) *C. graminicola*, (B) *C. destructivum* and (C) *C. tabacum*. The Y-axis legend on panel A applies to all panels likewise (note the different scale for *C. tabacum*). For each enzyme family, the bar is subdivided according to the number of enzymes identified. PAI: Protein Abundance Index.

748 **Figure 5.** Heatmap of the abundance of AA5 detected in the fungal secretomes.

749 **Figure 6: Comparative activity profiles of the three *Colletotrichum AA5_2* AlcOx, (A)**
750 Relative activity of the recombinant AlcOx measured by ABTS/HRP coupled assay on 3 mM
751 BnOH. (B) Relative activity profiling of the recombinant AlcOx enzymes on a set of substrates, determined by ABTS/HRP coupled assay, with activity on BnOH set as reference (activity on BnOH set as 100%). Substrate concentrations were as follows: 1% (w/v) xyloglucan, 50 mM for D-glucose, D-galactose, D-raffinose, 3 mM for butan-2-ol, butan-1-ol, benzyl alcohol, cinnamyl alcohol, octan-1-ol, decan-1-ol, and glycolaldehyde dimer. Reactions were performed with 1 nM of CgrAlcOx and CtaAlcOx, and 2 nM CdeAlcOx. Error bars show s.d. (independent experiments, n = 3).

758 **Figure 7: CgrAlcOx and CdeAlcOx mediated oxidation of some fatty alcohols. (A) Reaction** scheme proposed (based on (21)) (B) conversion of hexan-1-ol (C) Conversion of octan-1-ol. Reactions were incubated for either 15 minutes or 16 hours. All reaction mixtures contained: 3 mM substrate and 1 µM AlcOx, in phosphate sodium buffer (50 mM, pH 8.0) and 23°C. Reactions varied as follows: no accessory enzyme added, addition of catalase (8 µM final), addition of HRP (12 µM final), addition of both catalase (0.5 µM) and HRP (12 µM final). Legend in panel A applies for panel B likewise. Conversion products were analyzed by GC-FID. Error bars show s.d. (independent experiments, n = 3).

766
C. graminicola

YG
d7 d14

YG-CME
d7 d14

YG
d3 d7 d14

C. tabacum

YG-CME
d3 d7 d14

C. destructivum

YG
d3 d7 d14

<figure><img src="image_2.png" /></figure>
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>
A

C. graminicola

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

B

C. destructivum

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

C

C. tabacum

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>
<figure><img src="image_19.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_20.png" /></figure>
<figure><img src="image_21.png" /><figcaption>B</figcaption></figure>

C6

<figure><img src="image_22.png" /></figure>

CH3

<figure><img src="image_23.png" /></figure>

15 min

<figure><img src="image_24.png" /></figure>

AlcOx

+ Cat

AlcOx

+ HRP

AlcOx

+ Cat

+ HRP

AlcOx

<figure><img src="image_25.png" /></figure>

16 h

<figure><img src="image_26.png" /></figure>

CgrAlcOx

CdeAlcOx

<figure><img src="image_27.png" /></figure>

CgrAlcOx

CdeAlcOx

<figure><img src="image_28.png" /></figure>

CgrAlcOx

CdeAlcOx

<figure><img src="image_29.png" /></figure>

CgrAlcOx

CdeAlcOx

<caption>C</caption>

C8

<figure><img src="image_30.png" /></figure>

CH3

<figure><img src="image_31.png" /></figure>

15 min

<figure><img src="image_32.png" /></figure>

AlcOx

+ Cat

AlcOx

+ HRP

AlcOx

+ Cat

+ HRP

AlcOx

<figure><img src="image_33.png" /></figure>

16 h

<figure><img src="image_34.png" /></figure>

CgrAlcOx

CdeAlcOx

<figure><img src="image_35.png" /></figure>

CgrAlcOx

CdeAlcOx

<figure><img src="image_36.png" /></figure>

CgrAlcOx

CdeAlcOx

<figure><img src="image_37.png" /></figure>

CgrAlcOx

CdeAlcOx