Construction and Optimization of Engineered <i>Saccharomyces cerevisiae</i> for <i>De Novo</i> Synthesis of Phloretin and Its Derivatives

Phloretin and its derivatives are dihydrochalcone compounds with diverse pharmacological properties and biological activities, offering significant potential for applications in the food and pharmaceutical industries. Due to their structural similarity to flavonoids, their extraction and isolation were highly challenging. Although the biosynthesis of phloretin via three distinct pathways has been reported, a systematic comparison within the same host has yet to be conducted. In this study, we employed rational design and synthetic biology approaches to engineer <i>Saccharomyces cerevisiae</i> for <i>de novo</i> synthesis of phloretin and its derivatives. We constructed and evaluated three biosynthetic pathways for phloretin in <i>S. cerevisiae</i>, demonstrating that effective phloretin synthesis is achievable only via the <i>p</i>-coumaryl-CoA pathway. Additionally, by optimizing enzyme screening, strain engineering, and coordinating heterologous pathways with endogenous metabolism, we achieved the highest reported <i>de novo</i> titer of 287.2 mg/L for phloretin, 184.6 mg/L for phlorizin, 103.1 mg/L for trilobatin, and 164.5 mg/L for nothofagin and the first-time synthesis of 4-methylphloretin and hesperetin dihydrochalcone. This study was committed to addressing the growing demand for dihydrochalcones while laying the foundation for the biosynthesis of more complex derivatives.