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

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. 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.

Genome evolution and transcriptome plasticity associated with adaptation to monocot and eudicot plants inColletotrichumfungi

“…These enzymatic properties cannot be readily attributed to the lifestyle of these plant pathogens. Hydrogen peroxide (H2O2) may have multiple roles in plant pathogenic fungi because two subfamily AA5_2 alcohol oxidases have been characterized from C. graminicola and C. gloeosporioides 41,42 . These enzymes have broad substrate ranges and oxidize aliphatic primary alcohols to the corresponding aldehydes, by simultaneously reducing oxygen to hydrogen peroxide.…”

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity

“…The AA5_2 subfamily historically contains GalOx enzymes (EC1.1.3.9) [5,11,12] and has more recently included raffinose oxidases (RafOx; EC1.1.3.‐) [13], as well as other enzymes with varying substrate specificities, i.e. alcohol oxidases (AlcOx, EC1.1.3.13) [14–19] and aryl alcohol oxidases (AAO, EC1.1.3.7) [20,21]. Despite their differences in substrate specificities and modularities, AA5_2 enzymes share the same catalytic mechanism with the regioselective oxidation of a primary hydroxyl group into the corresponding aldehydes, with a concomitant reduction in dioxygen to hydrogen peroxide [22–24].…”

“…alcohol oxidases (AlcOx, EC1. 1.3.13) [14][15][16][17][18][19] and aryl alcohol oxidases (AAO, EC1.1.3.7) [20,21]. Despite their differences in substrate specificities and modularities, AA5_2 enzymes share the same catalytic mechanism with the regioselective oxidation of a primary hydroxyl group into the corresponding aldehydes, with a concomitant reduction in dioxygen to hydrogen peroxide [22][23][24].…”

Tunable Production of (R)- or (S)-Citronellal from Geraniol via a Bienzymatic Cascade Using a Copper Radical Alcohol Oxidase and Old Yellow Enzyme

“… 48 Only recently a few studies have started to investigate the characteristics and application potential of CRO-AlcOx. 25 , 49 − 51 A better understanding of these enzymes is needed to foster their use as biocatalysts. To date, CRO-AlcOx have never been evaluated for application in multistep enzymatic reactions, while alcohol oxidation is a key step in the synthesis route of many valuable chemicals.…”

“…CRO-AlcOx (EC 1.1.3.13; AA5_2 , ) are organic cofactor-free enzymes that recently emerged from the exploration of the fungal CROs family. ,, CROs are better known through the archetypal galactose 6-oxidase from Fusarium graminearum ( Fgr GalOx; EC 1.1.3.9; AA5_2), extensively studied, − engineered, − and broadly applied − since their initial discovery more than 60 years ago . Only recently a few studies have started to investigate the characteristics and application potential of CRO-AlcOx. ,− A better understanding of these enzymes is needed to foster their use as biocatalysts. To date, CRO-AlcOx have never been evaluated for application in multistep enzymatic reactions, while alcohol oxidation is a key step in the synthesis route of many valuable chemicals .…”