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

Abstract

There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine. Key points • Some environmental bacteria are able to use 5-KF as an energy and carbon source. • Four 5-KF reductases were identified, belonging to three different protein families. • Many gut bacteria cannot degrade 5-KF.

5-Keto-D-Fructose, a Natural Diketone and Potential Sugar Substitute, Significantly Reduces the Viability of Prokaryotic and Eukaryotic Cells

“…The search for substances that provide a pleasant and sufficient sweetening power without triggering the mentioned side effects led to the discovery of 5-keto- D -fructose (5-KF, 5-ketofructose, 5-oxofructose, 5-dehydro- D -fructose, threo-2,5-hexodiulose), a natural diketone found in honey, white wine, and vinegar in low concentrations ( Burroughs and Sparks, 1973 ; Schiessl et al, 2021 ). This potential sugar substitute can be produced by enzymatic oxidation of D -fructose, a reaction catalyzed by the membrane-bound fructose dehydrogenase complex (FDH SCL ) found in a variety of acetic acid bacteria ( Mowshowitz et al, 1974 ; Ameyama et al, 1981 ; Kawai et al, 2013 ).…”

“…This monophosphate was found to be a potent competitive inhibitor of the D -fructose-1-phosphate cleavage catalyzed by liver aldolase ( Englard et al, 1972 ). Concerning microbial degradation, it was demonstrated that 5-KF could not be degraded by 15 members of the most common and abundant intestinal microorganisms ( Schiessl et al, 2021 ). Thus, the sugar derivative seems to be an unsuitable growth substrate for prokaryotes in the human intestine.…”

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5‐ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase

“…As such, 5‐ketofuctose (5‐KF) is an interesting product. It is a naturally occurring compound that has a similar taste and sweetness as fructose (Herweg et al ., 2018 ), but is not metabolized by the human body (Wyrobnik and Wyrobnik, 2006 ) and, as recently shown, not metabolized by 15 prominent bacterial species of the human gut microbiome (Schiessl et al ., 2021 ). 5‐KF therefore meets crucial demands for a new sweetener.…”