Optimization of different membrane-bound dehydrogenases and their expression in Gluconobacter oxydans

Gluconobacter oxydans has great biotechnological potential due to its ability to incompletely oxidize many sugars, polyols, and related compounds with the preservation of their carbon skeletons. It can be used in fermentations with highly concentrated substrates and tolerates low pH. These oxidations are catalyzed by membrane-bound dehydrogenases (mDHs), with the active site facing toward the periplasm, circumventing the necessity to transport substrates and products. 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 platform was used to produce various chemicals of industrial importance. The membrane-bound glucose dehydrogenase (mGDH) and the membrane-bound polyol dehydrogenase (mSldAB) from G. oxydans are responsible for the production of sugar acids (cellobionic acid and galactaric acid) and ketoses (L-xylulose and D-tagatose) respectively. L-xylulose and D-tagatose are valuable in the food industry as low-calorie sweeteners and potential prebiotics, supporting health-conscious consumer trends. Cellobionic- and galactaric acid are important intermediates for biodegradable materials and pharmaceuticals, promoting sustainable practices in various manufacturing sectors.

To construct a strain that produces these sugar acids and ketoses with high space-time yields, a collection of BP9.1 strains expressing various homologous mGDH and mSldAB enzymes based on sequence and phylogeny was constructed. Additionally, new isolates from environmental samples were utilized as sources of novel mGDH and mSldAB enzymes, expanding our repertoire of biocatalysts. The expression was carried out using both plasmid-based systems and genes integrated into the chromosome of BP9.1.

For further optimization of enzyme activity, random mutagenesis is used to generate diverse enzyme variants, enabling the identification of mutants that enhance activity, specificity, and stability. This approach should increase the likelihood of obtaining improved enzymes for industrial needs. To facilitate the transfer of large libraries of mutated enzyme genes into BP9.1, we optimized and modified the triparental conjugation method to establish a high throughput protocol, significantly accelerating the screening process and enabling efficient selection of the most promising enzyme variants for industrial applications.