Metabolic Engineering of <i>Saccharomyces cerevisiae</i> for High-Level Production of <scp>l</scp>-Pipecolic Acid from Glucose

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain <i><i>Saccharomyces cerevisiae</i></i> BY4743. Initially, an optimized biosynthetic pathway was constructed within <i>S. cerevisiae</i>, converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask <i>in vivo</i>. Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.