<i>De Novo</i> Biosynthesis of ChlorogenicAcid in <i>Yarrowia lipolytica</i> through Cis-Acting ElementOptimization and NADPH Regeneration Engineering

Chlorogenic acid (CGA) is a natural hydroxycinnamic acid ester with significant applications in food preservation and nutritional health. However, extraction of CGA from plants is challenging, resulting in low purity that fails to meet increasing market demands. Furthermore, the broad substrate specificity of hydroxycinnamoyl-CoA:quinic acid transferase catalysis generating a plethora of byproducts, lack of NADPH regeneration, and the presence of degrading proteins impede microbial synthesis of CGA. This study achieved <i>de novo</i> synthesis of CGA in <i>Yarrowia lipolytica</i> by introducing hydroxylation and condensation modules based on screening synthetic pathway genes and optimizing parallel promoters. Additionally, an NADPH regeneration system was incorporated to enhance the efficiency of hydroxylation, thereby increasing the titer of CGA to 333.16 mg/L. From transcriptome data, 528 significantly upregulated genes were identified, and deletion of <i>YALI0_B21824g</i> significantly slowed the rate of CGA degradation, which increased the titer of CGA to 351.33 mg/L in shake flasks. Applying fed-batch fermentation in a 5 L bioreactor further increased CGA production to 4837.32 mg/L (64 mg/g DCW). This study established <i>de novo</i> synthesis of CGA in <i>Y. lipolytica</i>, providing a foundation for microbial production of coumaric acid and its derivatives.