RT Journal Article SR Electronic T1 Time-resolved secretome analysis of three Colletotrichum species identifies copper radical alcohol oxidases for the production of fatty aldehydes JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.06.09.447826 DO 10.1101/2021.06.09.447826 A1 David Ribeaucourt A1 Safwan Saker A1 David Navarro A1 Bastien Bissaro A1 Elodie Drula A1 Lydie Oliveira Correia A1 Mireille Haon A1 Sacha Grisel A1 Nicolas Lapalu A1 Bernard Henrissat A1 Richard J. O’Connell A1 Fanny Lambert A1 Mickaël Lafond A1 Jean-Guy Berrin YR 2021 UL http://biorxiv.org/content/early/2021/06/10/2021.06.09.447826.abstract AB Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. 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 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 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 as intermediates in preparative chemistry and for their use as flavors and fragrances ingredients. In the search for new biocatalysts, CRO-AlcOx have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. The use of crude fungal secretomes is often seen has an appealing approach by industries since alleviating various costs pertaining to biocatalysts production. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored. This study showed that Colletotrichum species can secrete a broad diversity of copper-containing enzymes, but only little amount of CRO-AlcOx. Thus, recombinant expression remains the most promising approach. The potential of CRO-AlcOx as biocatalyst for flavor and fragrance applications was confirmed through the production of two new enzymes with activity on fatty alcohols.