Modular RNAi Pathway Engineering Enhances Plasmid Copy Number Control in Yeast Bioproduction System

Rational design of microbial cell factories requires precise coordination of gene dosage and expression dynamics to optimize metabolic flux while minimizing cellular burden. In this study, an unexpected plasmid copy number amplification was identified following the reconstruction of an orthogonal RNA interference (RNAi) system in Saccharomyces cerevisiae, and a synthetic biology chassis with dynamically programmable plasmid copy numbers was developed. By integrating heterologous RNAi pathway genes from Saccharomyces castellii and designing sequence‐specific small interfering RNAs (siRNAs) targeting plasmid‐encoded selection markers, we established a chemically inducible gene dosage control platform capable of achieving plasmid copy number amplification up to 7.13‐fold. Application of this RNAi‐mediated copy number modulation to the carotenoid biosynthetic pathway resulted in an 18.6‐fold increase in lycopene titers compared to static plasmid systems. This study presents an innovative approach for dynamic plasmid copy number regulation in S. cerevisiae to enable high‐efficiency gene dosage control, further enriching the toolkit for synthetic metabolic regulation. This strategy exhibits significant potential for enhancing the production performance of microbial cell factories and offers novel perspectives for metabolic engineering optimizations within the synthetic biology framework.