Advances in Metabolic Engineering of L-Pipecolic Acid Supply in Tacrolimus Biosynthesis

Abstract

Tacrolimus (FK506), the first-line immunosuppressant for organ transplantation, is produced by fermentation of Streptomyces tsukubaensis. L-pipecolic acid constitutes the piperidine ring moiety of FK506; its synthase FkbL exhibits intrinsically low catalytic efficiency, representing the most flux-constrained step in the entire biosynthetic pathway. This review systematically compares recent strategies to overcome this bottleneck-including precursor pathway enhancement, competitive branch redirection, transcriptional and translational regulatory engineering, and fermentation optimization-with a particular focus on structure-guided enzyme design, multi-omics-driven translational optimization, and multi-target synergistic engineering. The current research shows that the hierarchical transcription control efficiency combining multi-copy fkbn and multi-gene cassettes is the best. "Structural Guide" engineering design and rare codon optimization are also new methods to increase production. The future research directions are FkbL directed evolution, the replacement of the whole rare codon, the discovery of indirect control factors, and the topic of simulated biosynthesis using CRISPR.