Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains
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The growing consumer demand for low-calorie, sugar-free foodstuff motivated us to search for alternative non-nutritive sweeteners. A promising sweet-tasting compound is 5-keto-d-fructose (5-KF), which is formed by membrane-bound fructose dehydrogenases (Fdh) in some Gluconobacter strains. The plasmid-based expression of the fdh genes in Gluconobacter (G.) oxydans resulted in a much higher Fdh activity in comparison to the native host G. japonicus. Growth experiments with G. oxydans fdh in fructose-containing media indicated that 5-KF was rapidly formed with a conversion efficiency of 90%. 5-KF production from fructose was also observed using resting cells with a yield of about 100%. In addition, a new approach was tested for the production of the sweetener 5-KF by using sucrose as a substrate. To this end, a two-strain system composed of the fdh-expressing strain and a G. oxydans strain that produced the sucrose hydrolyzing SacC was developed. The strains were co-cultured in sucrose medium and converted 92.5% of the available fructose units into 5-KF. The glucose moiety of sucrose was converted to 2-ketogluconate and acetate. With regard to the development of a sustainable and resource-saving process for the production of 5-KF, sugar beet extract was used as substrate for the two-strain system. Fructose as product from sucrose cleavage was mainly oxidized to 5-KF which was detected in a concentration of over 200 mM at the end of the fermentation process. In summary, the two-strain system was able to convert fructose units of sugar beet extract to 5-KF with an efficiency of 82 ± 5%.
KeywordsSweetener Fructose Sugar-free food Metabolic disorders Sugar beet Sugar substitute Incomplete oxidation Fructose dehydrogenase
This project was supported by funds from NRW-Strategy project BioSC (project GLUFACT) and from Bundesministerium für Bildung und Forschung (BMBF, project no. 031B0370A). We thank Elisabeth Schwab and Juliane Hoffmann for technical assistance, Dr. S. Kehraus of the Institute of Pharmaceutical Biology Bonn for the 13C NMR spectroscopy analysis, and Dr. P. Kasten of the Rheinischer Rübenbauer-Verband e.V. for the provision of sugar beet plants.
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