Aromatoleum aromaticum EbN1 and other species of the genus Aromatoleum have been sources for many enzymes catalyzing reactions with aromatic carbohydrates. Aromatic carboxylic acids can be found in various sources from crude oil to lignin. They have a broad range of applications. So far, industrial synthesis and applications rely on lithium/aluminium catalysts amongst others. Here we present a pathway way of biocatalytic reduction from aromatic acids to corresponding alcohols utilizing recombinant enzymes from A. aromaticum EbN1.

The tungsten cofactor containing aldehyde oxidoreductase (AOR) is an oxygen sensitive enzyme that is able to catalyze the oxidation of a broad variety of aromatic and aliphatic aldehydes to the respective acids in presence of an electron acceptor. [1] Recent results show that AOR is also catalyzing the reverse reaction, reduction of benzoate to benzaldehyde, albeit at very low rates and under conditions strongly favoring acid reduction, e.g., low pH. Reports of thermophilic AOR orthologs also demonstrate slow reduction of organic acids directly to the corresponding aldehydes, if the thermodynamic equilibrium is made favorable for this reaction by the presence of semicarbazide or alcohol dehydrogenases removing the aldehydes from equilibrium [2, 3] Therefore, introduction of a coupled system with a benzyl alcohol dehydrogenase (BADH) also from A. aromaticum EbN1 enabled us to study the kinetics for the acid reduction reactions of AOR.

It has been reported that BADH is produced in various growth conditions. [4] We established recombinant expression and purification of BADH to study its kinetics for reduction of aldehydes and oxidation of alcohols. Furthermore, the BADH itself has a broad substrate spectrum of mainly aromatic compounds and favors NAD(H) as cofactor, but works with NADP(H) as well. This allows multiple experimental settings, either including or excluding cofactor.

[1] Arndt, F. et al. (2019). Front. Microbiol. 10:71. doi: 10.3389/fmicb.2019.00071

[2] Heider, J. et al. (1995). J. Bacteriol. 177, 4757–4764. doi: 10.1128/jb.177.16.4757-4764.1995

[3] Huber, C. et al. (1995). Arch. Microbiol. 164, 110–118. doi: 10.1007/BF02525316

[4] Wöhlbrand, L. et al. (2007). Proteomics 7, 2222–2239. doi: 10.1002/pmic.200600987