Abstract:

Carbohydrates are essential bioresources with wide-ranging applications in the fields of functional foods, pharmaceuticals, energy, and advanced materials. The traditional methods for carbohydrate production, which typically rely on cellular systems, often suffer from inherent limitations such as low product yield, complex pathway regulation, and limited scalability. In recent years, in vitro biosynthesis technology has emerged as a groundbreaking alternative, offering a highly versatile, cell-free platform for the efficient synthesis of carbohydrates. This technology integrates multiple enzymes within a single reaction system, enabling them to work synergistically without the constraints of a living cell. This approach offers several significant advantages over traditional cellular synthesis. First, it bypasses the steady-state limitations of cellular systems, allowing for higher reaction rates and enhanced product yields. Second, it provides greater pathway controllability, making it possible to fine-tune reaction conditions for optimal performance. Third, it enhances the atomic economy, as the absence of cellular byproducts leads to more efficient use of substrates. These attributes make in vitro biosynthesis a powerful tool for the scalable and sustainable production of a wide range of carbohydrate products. This review provides a comprehensive overview of the core technological advancements in the in vitro carbohydrate synthesis. The discovery, engineering and expression of target enzymes are the foundation of the in vitro biosynthesis, providing the basic elements for pathway construction. For the pathway design, thermodynamic driving and cofactor recycling strategies ensure sustained catalytic activity and energy efficiency. Furthermore, the modular design of synthesis pathways allows for flexible configuration and rapid optimization of multi-enzyme systems, while synergistic adaptation mechanisms enable the efficient coordination of enzyme functions. This review also highlights representative applications of in vitro biosynthesis technology in the production of various monosaccharides, sugar alcohols, oligosaccharides, polysaccharides, and sugar derivatives, demonstrating its advantages in utilizing low-cost resources, achieving atomic economy, and enabling scalable production. Moreover, the potential of artificial intelligence to further enhance in vitro carbohydrate biosynthesis is discussed, particularly in the areas of enzyme discovery and modification, pathway optimization, and process parameter prediction. As the convergence of biology with advanced computational tools continues, in vitro carbohydrate biosynthesis is expected to accelerate its transition from laboratory research to industrial-scale applications, becoming a key enabler of next-generation green biomanufacturing.

Key words: carbohydrate resources, in vitro biosynthesis, enzyme engineering, pathway design, thermodynamic driving, cofactor recycling

摘要：

糖质是功能食品、医药、能源与材料领域中至关重要的生物资源。近年来，作为一种新兴的无细胞合成平台，体外生物合成技术正逐渐成为糖质制造的重要技术路径。该技术通过整合多种酶在单一反应体系中协同催化，突破了细胞底盘的稳态限制，具备更高的路径可控性、反应效率与原子经济性。本文系统回顾了糖质体外合成体系的核心技术进展，包括新酶挖掘与分子改造、热力学驱动与辅酶循环策略、模块化路径设计及多酶协同适配机制；同时梳理了该技术在单糖/糖醇、寡糖、多糖及糖衍生物等方面的代表性应用案例，展现其在廉价资源利用、原子经济性及规模化生产方面的优势。此外，本文还分析了人工智能在新酶挖掘改造、反应路径优化、工艺参数预测等糖质体外生物合成关键技术中的应用潜力。随着生物学与众多学科的深度交叉融合，糖质体外生物合成正加速从实验室走向工业化，有望成为下一代绿色生物制造的重要支撑。

关键词: 糖质资源, 体外生物合成, 酶工程, 路径设计, 热力学驱动, 辅酶循环