质粒拷贝数工程优化酿酒酵母中邻氨基苯甲酸甲酯的产量

Methyl anthranilate (MANT) is an aromatic compound with an orange blossom fragrance, widely used in industrial and agricultural applications. In this study, we developed a plasmid copy number engineering (PCNs) strategy to overcome the technical bottleneck of low genetic manipulation efficiency in Saccharomyces cerevisiae BJ5464-NpgA, and achieved significant enhancement of methyl anthranilate production using this engineered strain.Using red fluorescence intensity as an indicator of plasmid copy number, we precisely modulated the selection pressure by truncating the promoter of the Zeocin resistance gene and simultaneously elevating antibiotic concentration, thereby boosting plasmid copy number. The optimal parameters were determined to be a 30 bp truncated promoter under a Zeocin concentration of 300 mg/L.When applied to the heterologous synthesis of MANT in BJ5464-NpgA, HPLC analysis revealed that the engineered strain achieved a titer of 43.57 mg/L, compared to 15.51 mg/L in the control strain, representing a 1.81-fold increase in production. This strategy，based on an episomal plasmid expression system, provides a novel approach for optimizing heterologous metabolic pathways in Saccharomyces cerevisiae, and offers valuable insights for the development of microbial platforms for natural product biosynthesis.