not-yet-known not-yet-known not-yet-known unknown Violacein is a natural microbial purple pigment that is used as an easily screenable model compound in synthetic biology. In spite of its broader application potential as an antimicrobial, antiviral and antitumor agent, its low yield and the instability of wild-type production strains preclude its industrial production, while heterologous expression is also still at the laboratory scale. Therefore, more effective methods to synthesize violacein based on rational design have become a focus of research. In this study, the violacein biosynthesis pathway was reconstructed in Saccharomyces cerevisiae, but no production of violacein was initially observed. Analysis of the biosynthetic pathway revealed that low vioC expression was responsible for the low yield of violacein. Through pathway engineering, including multicopy integration and overexpression, followed by optimization of the fermentation medium via Box-Behnken design, the violacein yield in 250 mL shake flasks reached 143.89 mg/L, the highest value reported in S. cerevisiae to date. The heterologous reconstruction of the violacein biosynthesis pathway in S. cerevisiae presented here offers theoretical guidance for the de novo biosynthesis of this bioactive compound and related molecules.