Cell-free synthesis of high-order carbohydrates from low-carbon molecules.

Artificial conversion of CO2 into foods and chemicals offers a sustainable way to tackle population-related and environmental issues. Thermochemical and electrochemical methods for reducing CO2 to low-carbon molecules have made significant progress; however, it remains challenging to generate long-chain, structurally diverse carbohydrates, which are the most abundant substances in nature. Here, we report the successful design and realization of in vitro sucrose synthesis from C1 and C3 molecules through a thermodynamically favorable pathway with short reaction steps and low energy input. By engineering the phosphatase and sucrose synthase to improve their catalytic efficiency by 3- to 71-fold, and by optimizing the reaction system through an iterative scanning strategy, we achieved a high conversion yield of 86% and a specific synthetic rate of 5.7 g L-1 h-1. Additionally, we demonstrated the cell-free starch synthesis without the need for dextrin primers, achieving a superior C1-to-starch synthesis rate compared to previously developed multienzyme systems. Building upon this platform, we further extended methanol conversion to a variety of sugars, especially for cellooligosaccharides with a degree of polymerization of Cn≥18. Together, our system provides a promising, plant-independent route for de novo synthesis of structure-diversified oligosaccharides and polysaccharides.