초록
<P><B>Abstract</B></P> <P>Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates – glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An <I>in vitro</I> synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from <I>Thermotaga maritima</I>, phosphoglucomutase from <I>Thermococcus kodakarensis</I>, glucose 6-phosphate isomerase from <I>Thermus thermophilus</I>, and pyrophosphate phosphofructokinase from <I>T. maritima</I>) were overexpressed in <I>E. coli</I> BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70°C, respectively. The optimal enzyme ratios of αGP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 ± 4.6mM FDP was synthesized after 7-h reaction. This <I>de novo</I> ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A novel <I>in vitro</I> pathway was designed for the production of A high-energy metabolite without use of ATP. </LI> <LI> A set of hyperthermophilic enzymes were applied and the reaction conditions were optimized for high titers achieved. </LI> <LI> Cell-free catalysis led to higher reaction rate, higher product yield and easy product separation. </LI> </UL> </P>