Plasmid copy number (PCN) is a key factor limiting the expression level of heterologous proteins in yeast. Static strategies for enhancing PCN, such as reducing the transcriptional intensity of selection markers or increasing selection pressure, only maintain PCN at a single fixed level and struggle to achieve dynamic, precise, and reversible copy number regulation. This study established a dynamic plasmid copy number regulation strategy based on CRISPR interference (CRISPRi). Flexible control of PCN was achieved by designing specific guide RNAs (gRNAs) and integrating them into the inducible CRISPRi system. Optimization of the gRNA target site, inducer concentration, and induction timing resulted in a >2-fold increase in the fluorescence intensity of yeast-enhanced green fluorescent protein (yeGFP) compared with the group without induction. Using naringenin synthesis as proof-of-concept, this regulatory tool was applied to modulate the expression of chalcone synthase (CHS), the rate-limiting enzyme in naringenin biosynthesis. Finally, the yield of naringenin increased by 35.62% under the optimal induction conditions.
Xu et al. (Wed,) studied this question.