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. <i>Pichia pastoris</i>, 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 <i>P. pastoris</i>, we optimized the synthetic expression system (SES) by engineering heterologous core promoters derived from <i>Trichoderma reesei</i>. Through RNA-seq analysis, we identified high-expression genes' promoters from <i>T. reesei</i>, which were integrated into the SES system to improve its efficiency in <i>P. pastoris</i>. A total of 214 candidate promoters were synthesized and screened based on expression of mCherry, and 54 of them were found to function in <i>P. pastoris</i>. 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 <i>P. pastoris</i>. 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 <i>P. pastoris</i>.