Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4--sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5′ untranslated region, the N-terminal encoding sequence, and the chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

中文翻译：

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增强软骨素4-O-磺基转移酶的表达一锅酶法合成硫酸软骨素A

硫酸软骨素 (CS) 是治疗骨关节炎的膳食补充剂中的关键化合物，引发了人们对环境友好和安全的 CS 生产的生物技术追求的极大兴趣。 CS（例如CSA）的酶法合成被认为是最有前途的方法之一。然而，一直遇到的瓶颈是CSA生物合成过程中软骨素4-磺基转移酶（C4ST）的活跃表达。这项研究通过修改 5' 非翻译区、N 端编码序列和底盘，系统地增强转录、翻译和分泌机制，从而细致地深入研究了优化 C4ST 表达。最终，活性 C4ST 表达上升至 2713.1 U/L，惊人地增加了 43.7 倍。通过应用C4ST培养液上清液并集成3'-磷酸腺苷-5'-磷酸硫酸盐（PAPS）生物合成模块，我们构建了用于CSA生物合成的一锅酶系统，实现了高达97.0%的显着磺化度。 C4ST 表达的显着增强和工程化一锅酶合成系统的开发有望以可定制的磺化度加速大规模 CSA 生物合成。