Comprehensive Insights into the Production of Long Chain Aliphatic Aldehydes Using a Copper-Radical Alcohol Oxidase as Biocatalyst

Abstract

The oxidation of alcohols is a cornerstone reaction in chemistry, notably in the flavors and fragrances industry where long chain aliphatic aldehydes are major odorant compounds. In a context where greener alternatives are sought after, biocatalysis holds many promises. Here, we investigated the ability of the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) -an organic cofactor-free enzyme belonging to the copper-radical oxidases (CROs) class -to convert industrially-relevant long chain aliphatic alcohols. CgrAlcOx is a competent catalyst for the conversion of octan-1-ol, when supported by the accessory enzymes peroxidase and catalase. Detailed examination of the products revealed the occurrence of an overoxidation step leading to the production of carboxylic acid for some aliphatic aldehydes and benzaldehyde derivatives. The partition between aldehyde and acid products varied upon substrate properties (chain length and propensity to form geminal-diols), enzyme specificity, and could be tuned by controlling the reaction conditions. In silico analyses suggested an inhibitory binding mode of long chain aliphatic geminal-diols and a substrate-induced fit mechanism for a benzyl alcohol-derivative. By demonstrating their natural ability to perform long chain aliphatic alcohol oxidation, the present study establishes the potential of fungal CRO-AlcOx as promising candidates for the green production of flavors and fragrances compounds.

Time-resolved secretome analysis of threeColletotrichumspecies identifies copper radical alcohol oxidases for the production of fatty aldehydes

“…Carboxylic acid was not noticeable for hexan-1-ol but was observed after 16 hours of conversion of octan-1-ol only when HRP was present in the reaction. Indeed, this overoxidation to the acid was shown previously to be mediated by HRP in the case of fatty aldehydes > C6 (21). In the same study, we observed that in the case of benzaldehyde derivatives (i.e.…”

“…Conversion results showed that CdeAlcOx behave similarly to CgrAlcOx with all the tested substrates (Figure 6). As previously observed with CgrAlcOx (21,33), CdeAlcOx alone was unable to promote full alcohol conversion. However, when both accessory enzymes were added to the reaction mixture, full conversion was readily achieved in 15 minutes.…”

“…The copyright holder for this preprint this version posted June 10, 2021. ; https://doi.org/10.1101/2021.06.09.447826 doi: bioRxiv preprint 6 being the first, most studied, and to date, most active CRO-AlcOx, namely CgrAlcOx (18,21,33). By performing a tblastn 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.…”

“…Fungi were maintained on Potato Dextrose Agar (PDA) plates, grown at 23°C for seven days and stored at 4°C before utilization. The recombinant Pichia pastoris X33 strains containing CgrAlcOx gene (GenBank EFQ30446.1) was recovered from previous work (18,21). DNA cloning and transformation of P. pastoris X33 with codon-optimized genes (for expression in P. pastoris) encoding CdeAlcOx and CtaAlcOx was carried out as described previously (65).…”

“…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 6 being the first, most studied, and to date, most active CRO-AlcOx, namely CgrAlcOx (18,21,33). By performing a tblastn 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.…”