Abstract：
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-O-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 Komagataella phaffii 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.
摘要：
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，推动了对环保和安全CS生产的生物技术追求的浓厚兴趣。CS例如CSA的酶促合成被认为是最有前途的方法之一。然而,始终遇到的瓶颈是CSA生物合成过程中软骨素4-O-磺基转移酶（C4ST）的活性表达。本研究通过转录的系统增强来仔细研究优化C4ST表达，翻译,和通过5'非翻译区修饰的分泌机制，N端编码序列，和Komagataellaphafii底盘。最终,活动C4ST表达式升级到2713.1U/L，代表惊人的43.7倍增长。通过应用C4ST的培养液上清液并整合3'-磷酸腺苷-5'-磷酸硫酸盐（PAPS）生物合成模块，我们构建了一个用于CSA生物合成的一锅法酶系统，达到显著的磺化程度高达97.0%。C4ST表达的显着增强和工程化的一锅法酶合成系统的开发有望加快具有可定制磺化度的大规模CSA生物合成。
