Time-resolved secretome analysis of threeColletotrichumspecies identifies copper radical alcohol oxidases for the 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. 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.

A simple and direct ionic chromatography method to monitor galactose oxidase activity

“…32 Taking advantage of recombinant enzyme production pipelines, a large diversity of AA5_2 enzymes has been recently characterized. 9,10,33–36…”

“…32 Taking advantage of recombinant enzyme production pipelines, a large diversity of AA5_2 enzymes has been recently characterized. 9,10,[33][34][35][36] GalOx activity has historically been measured by indirect, spectrophotometric methods that rely on quantifying the coproduct H 2 O 2 using a coupled horseradish peroxidase (HRP)/ 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. 37 Despite widespread use, such assays have certain limitations due to the cross-reactivity of H 2 O 2 or the instability of the reporter molecule (ABTS cation radical).…”

A Lignocellulolytic Colletotrichum sp. OH with Broad-Spectrum Tolerance to Lignocellulosic Pretreatment Compounds and Derivatives and the Efficiency to Produce Hydrogen Peroxide and 5-Hydroxymethylfurfural Tolerant Cellulases

“…The transcriptome of the fungus Colletotrichum lupini showed upregulation of catalase and peroxidase genes in response to plant infection [114]. The secretome analysis of Colletotrichum graminicola, Colletotrichum tabacum, and Colletotrichum destructivum reported the expression of H 2 O 2producing copper radical-alcohol oxidase [115], suggesting the presence of peroxidases in the genome of Colletotrichum species.…”

Biocatalytic oxidation of fatty alcohols into aldehydes for the flavors and fragrances industry

“…It is only recently, with the advent of natural diversity exploration, notably rendered possible by the availability of abundant genomic data, that the true potential of CROs scope of action was unveiled (Andberg et al, 2017;Mathieu et al, 2020;Mollerup et al, 2019;Paukner et al, 2015Paukner et al, , 2014Yin et al, 2015). Remarkably, a subgroup of AA5_2 CROs, so-called CRO-AlcOx, appeared to be of particular relevance for fragrant aldehydes production (Forget et al, 2020;Oide et al, 2019;Ribeaucourt et al, 2021bRibeaucourt et al, , 2021aYin et al, 2015). These enzymes, mainly isolated from Colletotrichum species, all exhibit a similar substrate specificity profile and comparable efficiencies.…”

“…Importantly, CRO-AlcOx can also convert non-activated alcohols with increasing affinity towards longer alkyl chains (Ribeaucourt et al, 2021a;Yin et al, 2015). For instance, 1-hexanol and 1-octanol (3 mM) could be fully converted by the AlcOx from Colletotrichum graminicola (CgrAlcOx) and C. destructivum (CdeAlcOx) within 15 min and under mild conditions (23°C, in aqueous media) (Ribeaucourt et al, 2021b(Ribeaucourt et al, , 2021a. Despite stable/increasing catalytic efficiencies up to C10, conversion of C10 alcohol was only partial compared to C6-C8 (unpublished results; see Table S1), which might be the result of increasing insolubility of the substrate or stronger inhibition of CgrAlcOx by the hydrated form of the aldehyde (Scheme 1) (Forget et al, 2020;Ribeaucourt et al, 2021a).…”