Glucose-to-Starch Conversion by a Synthetic Enzymatic Cascade

Starch is a vital global commodity for food and industry, yet its agricultural production is unsustainable, relying on inefficient photosynthesis, arable land, and stable climates. Meanwhile, the annual production of ∼180 million tons of cellulose primarily from crop residues is largely wasted through incineration or landfilling. While enzymatic saccharification efficiently depolymerizes cellulose into glucose, its conversion to starch is fundamentally limited by the insufficient catalytic efficiency, poor substrate selectivity of key enzymes, and the reversibility of the core reactions they catalyze. Here, we optimized a cell-free three-enzyme cascade, designated the carbon-economical starch synthesis (CESS) pathway, to address this limitation. This in vitro cascade integrates three core enzymes: polyphosphate glucokinase (PPGK) for adenosine triphosphate (ATP)-free glucose activation, phosphoglucomutase (PGM), and α-glucan phosphorylase (αGP). We engineered the rate-limiting αGP from Thermotoga petrophila to generate the M7 variant ( Tp αGPM7), achieving a 4.54-fold higher catalytic efficiency, and optimized cofactors, identifying 20 mmol∙L −1 Mn 2+ to critically shift PGM’s preference toward starch synthesis. The optimized pathway converts 5.4 g∙L −1 glucose (equivalent to 30 mmol∙L −1 ) to 3.50 g∙L −1 starch at 60 ℃ with a record-breaking 71.93% conversion yield and a space-time yield of 2.33 g∙L −1 ∙h −1 , while minimizing carbon loss < 30%. The product was characterized as amylose. This photosynthesis-independent framework advances a sustainable route to valorize cellulosic waste into food-grade starch, offering a resilient strategy for food security and a circular bioeconomy.