CRISPR-based synthetic translational regulation using non-conventional yeast

Abstract

Efficient and programmable gene expression systems are essential for improving recombinant protein production in non-conventional yeast hosts such as Pichia pastoris. In this thesis, a synthetic gene regulation platform was established in P. pastoris by integrating rationally engineered GAP promoters with CRISPR/dCas9-based transcriptional activation modules. The aim was to convert the native constitutive GAP promoter into a tunable element capable of both activation and repression through gRNA-guided recruitment of effector domains, thus paving the way for orthogonal and context-specific control of gene expression. Two synthetic promoter variants (version 1 and version 2) were designed by introducing targeted mutations to create novel gRNA binding sites without disrupting core promoter function. These promoters were cloned upstream of an eGFP reporter and integrated into the genome of P. pastoris. Colony screening under various carbon sources (glucose, glycerol, ethanol, and methanol) ii revealed that most mutant promoters retained expression levels comparable to the wild-type PGAP, while certain clones displayed elevated eGFP production due to multiple gene integrations. Quantitative PCR analysis was employed to identify single-copy integrants for further use. Subsequently, a CRISPRa system comprising dCas9, MS2-binding scaffold RNAs, and the VP64 activation domain was introduced into selected single-copy clones. Ten custom-designed gRNAs (five for each promoter version) were tested under four carbon conditions to assess their activation potential. Notably, version 1 demonstrated robust transcriptional activation with specific gRNAs, especially v1-g2-c1, which significantly enhanced eGFP expression across all tested conditions. In contrast, version 2 failed to elicit notable activation, possibly due to unfavorable gRNA positioning or inhibitory mutations within the promoter sequence. This work introduces a modular and orthogonal transcriptional regulation system in P. pastoris, offering dynamic control over synthetic promoters using CRISPRa components. The approach establishes a foundation for future metabolic engineering strategies and recombinant protein expression systems that are independent of traditional inducible promoters and adaptable to various industrial contexts.