Enhanced chlorogenic acid production from glucose via systematic metabolic engineering of Saccharomyces cerevisiae

Chlorogenic acid (CGA) is a valuable phenolic acid with various pharmaceutical functions. In our previous study, <i>de novo</i> synthesis of CGA in <i>Saccharomyces cerevisiae</i> was achieved. However, its yield required improvement before large scale production. In this study, systematic metabolic engineering strategy was used to reconstruct chassis cell <i>S</i>. <i>cerevisiae</i> YC0707 to enhance its CGA yield from glucose. To balance the supply of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), <i>ZWF1</i> (encoding glucose-6-phosphate dehydrogenase) and <i>GND1</i> (encoding 6-phosphogluconate dehydrogenase) were overexpressed by strong promoter <i>P</i> _<i>TEF1</i> swapping, thereby strengthening the pentose phosphate pathway. The mutant of phosphofructokinase (<i>PFK2</i> ^<i>S718D</i> ) was further introduced to weaken the glycolytic pathway. Then, the <i>p</i>-coumaric acid synthesis capacity was enhanced by employing tyrosine ammonia lyase from <i>Rhodotorula glutinis</i> (RgTAL), ΔHAM1, and ΔYJL028W. Fusion expression of AtC4H (cinnamate-4-hydroxylase) and At4CL1 (4-coumarate CoA ligase 1), together with CsHQT (hydroxycinnamoyl CoA quinate transferase) and AtC3'H (<i>p</i>-coumaroyl shikimate 3-hydroxylase), improved biosynthetic flux to CGA. Subsequently, the microenvironment of P450 enzymes was improved by overexpressing <i>INO2</i> (a transcription factor for lipid biosynthesis) and removal of heme oxygenase gene <i>HMX1</i>. Furthermore, screening potential transporters to facilitate CGA accumulation. Finally, we optimized the fermentation conditions. Using these strategies, CGA titers increased from 234.8 mg/L to 837.2 mg/L in shake flasks and reached 1.62 g/L in a 5-L bioreactor, representing the highest report in <i>S. cerevisiae</i> and providing new insights for CGA production.