ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering

- A novel in vitro pathway was designed for the production of A high-energy metabolite without use of ATP.
- A set of hyperthermophilic enzymes were applied and the reaction conditions were optimized for high titers achieved.
- Cell-free catalysis led to higher reaction rate, higher product yield and easy product separation.

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 in vitro 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 Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli 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 ± 4.6 mM FDP was synthesized after 7-h reaction. This de novo 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.