Metabolic Engineering\nof Saccharomyces\ncerevisiae for High-Level Production of l‑Pipecolic Acid from Glucose

l-Pipecolic acid (L-PA), an essential chiral\ncyclic nonprotein\namino acid, is gaining prominence in the food and pharmaceutical sectors\ndue to its wide-ranging biological and pharmacological properties.\nHistorically, L-PA has been synthesized chemically for commercial\npurposes. This study introduces a novel and efficient microbial production\nmethod for L-PA using engineered strain <i>Saccharomyces\ncerevisiae</i> BY4743. Initially, an optimized\nbiosynthetic pathway was constructed within S. cerevisiae, converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL\nshake flask <i>in vivo</i>. Subsequently, a multifaceted\nengineering strategy was implemented to enhance L-PA production: substrate-enzyme\naffinity modification, global transcription machinery engineering\nmodification, and Kozak sequence optimization for enhanced L-PA production.\nApproaches above led to an impressive 8.6-fold increase in L-PA yield,\nreaching 5.47 g/L in shake flask cultures. Further scaling up in a\n5 L fed-batch fermenter achieved a remarkable L-PA concentration of\n74.54 g/L. This research offers innovative insights into the industrial-scale\nproduction of L-PA.