Biotechnologische Anwendungen der Aldehyd‑Oxidoreduktase und anderer Enzyme aus Aromatoleum aromaticum

Gemmecker, Yvonne

Thesis

Open Access

Biotechnologische Anwendungen der Aldehyd‑Oxidoreduktase und anderer Enzyme aus Aromatoleum aromaticum

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Date

2025-11-27

Authors

Gemmecker, Yvonne

Abstract

The aldehyde:ferredoxin oxidoreductase from the beta-proteobacterium Aromatoleum aromaticum EbN1 (AORAa) is an enzyme with a three-subunit "nanowire" structure that catalyzes the redox reactions of various aldehydes and their corresponding carboxylic acids. The β subunits each contain a tungsten-bis-pterin cofactor in the active site, which is connected via a tunnel of [4Fe-4S] clusters in the β and α subunits to FAD cofactors in the γ subunits. In addition to the three structural genes aorA–aorC, two further genes in the operon, aorD and aorE, which encode maturation factors, are essential for AORAa production. Particularly notable in the enzyme’s architecture is the three-dimensional “nanowire” multimer complex. Structural analysis identified an α-helical region in AorA that is suspected to be responsible for oligomerization. A deletion mutant of this AorA, AorΔhelix, was constructed. It is kinetically characterized in this work and compared with the AORAa wild type. In vitro, AORAa activity can be measured with viologen dyes, Ti(III)-citrate, and NAD(H), although so far only NAD(H) is considered a physiological electron acceptor. In this study, it was tested whether ferredoxin could serve as a natural electron donor. In addition, phosphite, hypophosphite, methylene blue, and DCPIP were tested as electron donors or acceptors. Together with various alcohol dehydrogenases, the reduction of aromatic and aliphatic carboxylic acids by AORAa can continue to the corresponding alcohols. In addition to AORAa, A. aromaticum EbN1 harbours other enzymes of interest for this purpose. AdhB and BaDH are alcohol dehydrogenases that possess properties relevant for biocatalysis, such as tolerance to common solvents (AdhB, BaDH) and a broad substrate range for aromatic compounds (BaDH). Both enzymes have been characterized at a basic level. Notably, recombinant preparations of both proteins contain indications of a novel nucleotide-like cofactor. In combination with AORAa, they have also been used to reduce benzoate or acetic acid to the corresponding alcohols. The genome of A. aromaticum EbN1 contains the gene for another tungsten enzyme, AOR1. Although this type of enzyme occurs in other organisms, no representative has been characterized to date. In the course of this work, AOR1 was produced recombinantly and initially characterized. Optimizing growth conditions for various Aromatoleum spp. strains is not only important for a future biocatalytic system but has also contributed to the development of methods for the genetic manipulation of A. evansii. Lastly, it was shown that various members of the Aromatoleum group can use substances derived from lignin as a carbon source and that substrates for AORAa are present in lignin derivatives.