Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains

Abstract

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%.

Degradation of the low-calorie sugar substitute 5-ketofructose by different bacteria

“…The model organism G. oxydans 621H does not possess the key enzyme Fdh for 5-KF production (Siemen et al 2018 ). Therefore, we tested a fdh knock-in mutant of G. oxydans (Hoffmann et al 2020 ) for the expression of the fdh genes that allowed the production of the fructose dehydrogenase.…”

“…Moreover, there is no artificial, bitter or licorice-like aftertaste, which is noticeable when using some other alternative sweeteners such as stevia, saccharin, and acesulfame K (Soejarto et al 1982 ; Herweg et al 2018 ; Kuhn et al 2004 ). In addition, 5-KF is a natural sugar derivative that is found, e.g., in honey, white wine, and vinegar (Siemen et al 2018 ), leading to the question of how 5-KF is degraded in nature.…”

“…Some of the organisms, such as Gluconobacter ( G. ) japonicus , possess the enzyme fructose dehydrogenase, which can oxidize fructose to 5-KF (Ameyama et al 1981 ; Yamada et al 1966 ). The genes coding for fructose dehydrogenase were introduced into G. oxydans 621H (Siemen et al 2018 ) and the resulting genetically modified strain was capable of producing 5-KF in a fed-batch fermentation process resulting in product titers of up to 490 g L −1 and product yields up to 98% (Herweg et al 2018 ). Furthermore, a G. oxydans strain was developed for the efficient production of 5-KF from the cost-efficient and renewable feedstock sucrose (Hoffmann et al 2020 ).…”

Identification of NAD-Dependent Xylitol Dehydrogenase from Gluconobacter oxydans WSH-003

“…5 In particular, Gluconobacter oxydans has applications in the production of food additives and sweeteners owing to its ability to synthesize flavoring ingredients from aromatic alcohols, aliphatic alcohols, and 5-ketofructose. 6 Besides, G. oxydans enzymes, cell membranes, and whole cells are also used as sensor systems for the detection of polyols, sugars, and alcohols. 7 In recent years, some G. oxydans strains have been employed for the production of enantiomeric pharmaceuticals and platform compounds; for example, G. oxydans DSM2343 has been employed for the reduction of various ketones used in pharmaceutical, agrochemical, and natural products.…”

Identification of NAD-dependent xylitol dehydrogenase fromGluconobacter oxydansWSH-003

“…Gluconobacter oxydans has applications in the production of food additives and 67 sweeteners owing to its ability to synthesize flavoring ingredients from aromatic 68 alcohols, aliphatic alcohols, and 5-ketofructose (6,7). Besides, G. oxydans enzymes, 69 cell membranes, and whole cells are also used as sensor systems for the detection of 70 polyols, sugars, and alcohols (8)(9)(10).…”