Ultra-rapid and high-titer biomanufacturing of trehalose 6-phosphate by an in vitro synthetic biology platform.

Trehalose 6-phosphate (T6P), often referred to as plant insulin, serves as a central signaling molecule that regulates carbon partitioning and sucrose flux in plants, thereby influencing key agronomic traits, such as grain yield and drought resilience. Foliar spraying of T6P has been shown to significantly enhance yield and stress tolerance of numerous grains and vegetables. To circumvent the dependency on coenzymes in natural T6P synthesis, we designed and validated an in vitro new-to-nature, coenzyme-free, minimal enzymatic pathway for the biosynthesis of T6P from maltose and polyphosphate. This three-enzyme cocktail contained maltose phosphorylase, trehalose 6-phosphate phosphorylase, and polyphosphate glucokinase, and did not involve any costly coenzymes, such as ATP or UDP. Through systematic optimization of experimental parameters (including pH, temperature, Mg²⁺, phosphate concentration, and enzyme ratios), a 93% molar yield of T6P was achieved from 10 g/L maltose. The scale-up of this in vitro bioprocess to a 100-mL bioreactor with 200 g/L maltose enabled the production of up to 541 mM T6P (i.e., 252 g/L T6P disodium salt) within two hours, corresponding to a very high volumetric productivity of 126 g/L/h. This study established a scalable and cost-competitive in vitro biomanufacturing of T6P. Chemical intervention based on timed foliar spraying of T6P to cultivated crops offers a simpler and safer agricultural practice compared to genetic modification of crops.