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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. [Display omitted] [ABSTRACT FROM AUTHOR]