Characterization of Gluconobacter oxydans strains and development of a production process for 5-Ketofructose

  • Charakterisierung von Gluconobacter oxydans Stämmen und Entwicklung eines Prozesses zur Produktion von 5-Ketofructose

Herweg, Elena Ruth; Büchs, Jochen (Thesis advisor); Deppenmeier, Uwe (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2019

Abstract

Gluconobacter oxydans is known for its direct oxidations with membrane-bound dehydrogenases, but the bioprocess development is often challenging due to low biomass yields. To approach this difficulty the glucose metabolism of G. oxydans was genetically modified. Within this thesis two newly generated strains, as well as the reference strain 621H delta-upp were characterized regarding respiration activity and product formation. Two growth phases were identified in shake flask cultivations of the reference strain for oxidation of glucose to gluconate and further oxidation to 2-ketogluconate. In continuous cultivations, direct oxidation of glucose to gluconate was dominant at high dilution rates, D>0.3 h-1, whereas smaller dilution rates stimulated oxidation of gluconate to 2-ketogluconate, acetate production and an increased biomass formation. In the newly generated G. oxydans strains the direct oxidation of glucose was prevented and glucose utilization was improved by deletion of the genes for soluble and membrane-bound glucose dehydrogenases and the pyruvate decarboxylase. Genes for enzymes missing in the citrate cycle and genes for glucose uptake were inserted instead. Growth of the two new strains Mut1 and Mut2 was retarded in comparison to the reference strain, but gluconate formation was avoided. Optical density and CO2 formation was increased in both strains, indicating an increased carbon catabolism and biomass formation. A fed-batch process for the production of the potential sweetener 5-ketofructose (5-KF) from fructose with a recombinant G. oxydans strain overexpressing a fructose dehydrogenase from G. japonicus was developed. 5-KF production of the strain was characterized in shake flasks with online monitoring of the respiration activity and successfully scaled up to a 2 L fermentation. Fructose consumption rates were determined and suitable feed rates for fermentations with constant fructose feed were calculated. Up to 489 g/L 5-KF were produced in fed-batch fermentations with constant feeding and a product yield of 0.98 g5-KF/gfructose and a space time yield of 8.6 g/L/h were achieved. Mass balancing of the fermentations was conducted and the stoichiometry of the fermentations was determined. 5-KF was also produced with resting cells and purified with activated carbon to obtain a pure 5-KF solution without media supplements. All in all, an efficient fed-batch process for the production of 5-KF was developed.

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