Exploring Cell-Free Systems for Enhanced Synthesis of Hyaluronic Acid

作者：Zhi-Yuan Yao, Jin‐Song Gong, Xiongyan Yang, Jiancheng Shen, Minjun Yu, Chang Su, Heng Li, Guoqiang Xu, Shouyong Jiang, Frank Hollmann, Zhenghong Xu, Jin‐Song Shi · 发表于：ACS Sustainable Chemistry & Engineering · 年份：2025 · DOI：10.1021/acssuschemeng.5c08932 · 被引用次数：2 · 研究领域：Proteoglycans and glycosaminoglycans research、Carbohydrate Chemistry and Synthesis、Glycosylation and Glycoproteins Research

Hyaluronic acid (HA), a linear polysaccharide composed of alternating β-1,3-glucuronic acid (GlcA) and β-1,4- N -acetylglucosamine (GlcNAc) disaccharide units, is widely utilized in food, pharmaceutical, and cosmetic industries. Conventional in vitro HA biosynthesis is hindered by the reliance on costly nucleotide sugar precursors (UDP-GlcA and UDP-GlcNAc) and inefficient multienzyme coordination. To address these challenges, this study established a cell-free enzymatic cascade system integrating HA de novo synthesis with nucleotide recycling through eight pathway enzymes. By leveraging nucleotide sugar salvage pathways, UDP-GlcA and UDP-GlcNAc were efficiently synthesized from inexpensive monosaccharides, thereby bypassing energy-intensive de novo routes. Soluble expression of Pasteurella multocida hyaluronan synthase ( Pm HAS) was achieved by truncating its membrane-associated domains to enable sequential glycosyl transferase activity in aqueous systems. A dual ATP/UTP regeneration strategy was further implemented to sustain nucleotide supply, eliminating costly downstream purification. Under optimized conditions, the system produced 1.28 g/L HA within 24 h, with a molecular weight range of 1.28 × 10 4 to 1.02 × 10 6 Da and a substrate conversion yield of 65.9%. This work not only provides an economical platform for scalable HA synthesis but also establishes a modular enzymatic blueprint for engineering complex biopolymers, demonstrating broad applicability in synthetic gly...