Production of rare sugars by transketolase in combination with ketose 3-epimerase

Transketolase is an enzyme of the pentose phosphate pathway, and catalyzes the transfer of a 2-carbon unit from d-xylulose-5-phosphate to d-ribose-5-phosphate, to form d-sedoheptulose-7-phosphate (7-carbon ketose). Transketolase can also recognize non-phosphorylated monosaccharides as substrates, to form non-phosphorylated 7-carbon ketoses (heptuloses). In nature, monosaccharides are usually composed of 3-6 carbons, and higher carbon sugars composed of 7 or more carbons are rare. The higher carbon sugars may exhibit unknown, interesting bioactivity, and a method for their mass production should be established. Transketolase can form a C-C covalent bond, and is expected to show marked potential as a biocatalyst to synthesize higher carbon sugars. We carried out determinations of the optimal pH and temperature on enzyme activity, and X-ray structure determination of Thermus thermophilus HB8 transketolase (TtTK), and revealed that TtTK can efficiently synthesize four heptuloses: d-sedoheptulose, l-glucoheptulose, d-idoheptulose, and l-galactoheptulose, from lithium hydroxypyruvate and non-phosphorylated aldopentoses, d-ribose, l-arabinose, d-xylose, and l-lyxose, respectively. Transketolase is stereospecific and enantioselective to hydroxyaldehydes substrates (Cn) with an (2R) configuration resulting in the formation of d-threo (3S,4R) Cn+ 2 ketoses. Pseudomonas cichoriid-tagatose 3-epimerase (PcDTE) is a ketose 3-epimerase with a broad substrate specificity, and it was used for the production of a rare sugar, d-allulose from d-fructose (conversion rate: 25 %, purity: 99.5 %). We also clarified that PcDTE can successfully catalyze the epimerization at the 3-position of the four heptuloses obtained by TtTK, to give the four C3-epimers: d-alloheptulose, l-mannoheptulose, d-guloheptulose, and l-taloheptulose. These results demonstrate the potential of TtTK and PcDTE as enzymes producing heptuloses.