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(−)‐vibo‐Quercitol (VQ: 1L‐1,2,4/3,5‐cyclohexanepentol), a form of deoxyinositol, is an alternative chiral building block in the synthesis of bioactive compounds to control diabetes. In this study, an adenosine triphosphate‐free in vitro synthetic enzymatic biosystem composed of five enzymes (including one enzyme for NADH regeneration) was constructed to produce VQ from maltodextrin in one‐pot. After optimization of reaction conditions, 7.6 g/L VQ was produced from 10 g/L maltodextrin with a product yield (mol/mol) of 77%, and 25.3 g/L VQ with a purity of 87% was produced from 50 g/L maltodextrin through simple scaling up of this nonfermentative enzymatic biosystem. Therefore, this study provides an economical and environmentally friendly method for the envisioned quercitol biosynthesis. [ABSTRACT FROM AUTHOR]

Phosphatases, which catalyze the dephosphorylation of compounds containing phosphate groups, are important members of the haloacid dehalogenase (HAD)–like superfamily. Herein, a thermostable phosphatase encoded by an open reading frame of Trd_1070 from Thermomicrobium roseum was enzymologically characterized. This phosphatase showed promiscuous activity against more than ten sugar phosphates, with high specific activity toward ribose 5-phosphate, followed by ribulose 5-phosphate and fructose 6-phosphate. The half-life of Trd_1070 at 70 °C and pH 7.0 was about 14.2 h. Given that the catalytic efficiency of Trd_1070 on fructose 6-phosphate was 49-fold higher than that on glucose 6-phosphate, an in vitro synthetic biosystem containing alpha-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, and Trd_1070 was constructed for the production of fructose from maltodextrin by whole-cell catalysis, resulting in 21.6 g/L fructose with a ratio of fructose to glucose of approximately 2:1 from 50 g/L maltodextrin. This in vitro biosystem provides an alternative method to produce fructose with higher fructose content compared with the traditional production method using glucose isomerization. Further discovery and enzymologic characterization of phosphatases may promote further production of alternative monosaccharides through in vitro synthetic biosystems. [ABSTRACT FROM AUTHOR]

The regeneration of adenosine triphosphate is crucial for establishing a cost‐effective biosystem in which energy‐dependent processes are involved. Here, an in vitro synthetic enzymatic biosystem was designed to utilize low‐cost starch‐based material as energy source for the stoichiometric regeneration of ATP. This system required neither NAD(P) nor coenzyme A, and theoretically produced 3 ATPs per glucose equivalent of starch. When integrated with the L‐theanine‐producing enzymatic reaction which consumes 1 ATP to generate 1 L‐theanine molecule, our system resulted in a near‐theoretical yield of 2.94 L‐theanine per glucose equivalent. Furthermore, inorganic phosphate concentration of the system was nearly constant throughout the reaction period, suggesting the homeostatic reaction environment for enzymatic processes. This work provides a useful strategy for cost‐effective ATP regeneration which facilitates the production of ATP‐dependent value‐added chemicals. Not at all starchy! A designed NAD(P)/CoA‐free and phosphate‐balanced in vitro synthetic enzymatic biosystem allows for the stoichiometric regeneration of ATP from starch‐based material. 3 molecules of ATP are regenerated from each glucose equivalent of starch. [ABSTRACT FROM AUTHOR]

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis , was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli , and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D -tagatose 6-phosphate, and was relatively specific for this substrate than for D -glucose 1-phosphate, D -glucose 6-phosphate, and D -fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D -tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D -tagatose in the future. • We examined the enzymatic properties of a putative phosphatase from M. marburgensis. • This enzyme was thermostable and relatively specific for D -tagatose 6-phosphate. • This enzyme was applied in an in vitro synthetic biosystem to produce D -tagatose. [ABSTRACT FROM AUTHOR]