Engineering heterologous core promoters to optimize the SES expression system for Pichia pastoris

Synthetic biology has revolutionized microbial production by broadening the range of substrates, products, and host organisms. Pichia pastoris, a pivotal system for industrial enzyme production and biopharmaceuticals, encounters limitations in production capacity and transcriptional regulation, which impede its wider application. To enhance the performance of P. pastoris, we optimized the synthetic expression system (SES) by engineering heterologous core promoters derived from Trichoderma reesei. Through RNA-seq analysis, we identified high-expression genes' promoters from T. reesei, which were integrated into the SES system to improve its efficiency in P. pastoris. A total of 214 candidate promoters were synthesized and screened based on expression of mCherry, and 54 of them were found to function in P. pastoris. Detailed characterization based on fluorescence intensity identified 41 SES systems showing enhanced mCherry expression compared to the traditional SES-A by up to 5.4 times. The best SES-CP32 was successfully used to expresse Protease K (Pro K) in P. pastoris. Additionally, we employed a multi-copy strategy, utilizing CRISPR/Cas9 for rapid multi-targeted integration, to further enhance the expression efficiency of the SES system in producing Pro K, with the enzyme activity in the fermentation supernatant of 3 copy of Pro K being 4.6 times greater than 1 copy of Pro K. This study enhances the robustness and versatility of the SES system, establishing a foundation for expanding the utility of the SES system in P. pastoris.