Engineering <i>Pichia pastoris</i> as a Heterologous Platform for Non-Ribosomal Peptide Biosynthesis of the β-Lactam Intermediate δ-( <scp>l</scp> -α-Aminoadipyl)- <scp>l</scp> -cysteinyl- <scp>d</scp> -valine (ACV)

The production of nonribosomal peptides (NRPs) in non-native hosts remains challenging due to the size and complexity of the biosynthetic enzymes. Here, we report the first successful reconstruction of the δ-(L-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) pathway in Pichia pastoris. By introducing codon-optimized versions of the pcbAB and npgA genes directly into the genome, we were able to rebuild the minimal pathway required for ACV synthesis, which we confirmed using LC–MS/MS. Although the starting strain produced only small amounts of ACV (∼5 ng/mL), yields increased 10-fold after adaptive evolution (∼50 ng/mL) and further improved with precursor supplementation (∼60 ng/mL). Transcriptomic analysis showed that the engineered strain reorganized its metabolism to meet the energy and precursor demands of NRPS activity. Overall, this study suggests that P. pastoris can serve as a practical, proof-of-concept host for complex NRP biosynthesis and lays the groundwork for future engineering of β-lactam pathways.