Gluconobacter oxydans has great biotechnological potential due to its ability to incompletely oxidize a large variety of sugars, polyols, and related compounds. These oxidations are catalyzed by membrane-bound dehydrogenases (mDHs) with the active site facing toward the periplasm, therefore avoiding the transport of substrates and products in and out of the cytoplasm. We developed a platform for the functional expression of heterologous mDHs in G. oxydans BP9.1, devoid of its native mDHs, thereby increasing the specific activity of the heterologous mDHs and avoiding unwanted side reactions.

This study aims to use this platform to produce cellobionic acid (CBA), galactaric acid (GA) and other relevant sugar acids. The membrane-bound glucose dehydrogenase (mGDH) from Pseudomonas taetrolens was expressed in G. oxydans BP9.1 to construct a strain that produces CBA and GA with a high space-time yield. For further optimization, random in vitro mutagenesis was used to create variants of this enzyme. An assay was established to screen for mutants with a high oxidation rate. Carbonate in the medium serves as an indicator for acidification. By oxidizing the substances in the medium, the colonies form halos on the plates, whose size correlates with the amount of acid produced.

One common limitation of microbial processes using acetic acid bacteria compared to chemical processes is the relatively low space-time yield due to small cell concentrations, which makes industrial biomass production for oxidative biotransformation costly. Consequently, the opportunity to increase the growth yield could enhance productivity and result in improved profitability for industrial use. The non-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of acetic acid bacteria. It provides electrons to the electron transport chain, leading to the synthesis of ATP. Since BP9.1 seems to be limited in NADH dehydrogenase activity, its overexpression leads to a significantly increase in biomass formed. The presence of an additional NDH-2 in our platform strain enables more effective utilization of the generated NADH for energy production.