CRISPR-Mediated rDNA Integration and Fluorescence Screening for Pathway Optimization in Pichia pastoris

Gene dosage amplification is an effective strategy to improve the performance of heterologous genes and pathways. Pichia pastoris is an excellent recombinant protein expression host with high efficiency in protein folding and glycosylation. However, the traditional iterative multicopy integration method typically faces challenges such as being time-consuming and having high cost and potential gene mutations. Accordingly, we established CRISPR-mediated rDNA integration and fluorescence screening for pathway optimization (CRISPO) for multicopy pathway integration in a single-step and antibiotic-free manner. With geraniol biosynthesis as a case study, we designed CRISPO based on the use of glycerol-induced and glucose-repressed promoters (CRISPOi) or strong constitutive promoters (CRISPOc) to drive the expression of the red fluorescent protein mCherry as the screening marker. We employed CRISPOi for stable strain construction by multicopy integration of the geraniol synthase encoding gene, achieving a 19.5-fold increase in geraniol production. We demonstrated CRISPOc for visualizing and determining the rate-limiting steps of the mevalonate pathway, with HMG1 and ERG12 identified as the major rate-limiting enzymes through two rounds of exploration. Ultimately, CRISPO enabled us to construct an engineered P. pastoris strain producing 1.66 g/L geraniol (with a total of 2.12 g/L monoterpenoids) and 6.27 g/L geraniol (with a total of 6.48 g/L monoterpenoids) in 24-well plates and 5 L fermenters, respectively, representing the highest titer and productivity of geraniol ever reported. CRISPO is an important addition to the synthetic biology toolbox for the construction and optimization of P. pastoris cell factories.