Akademik Veri Yönetim Sistemi
11th Conference on Recombinant Protein Production, Girona, İspanya, 17 - 19 Ekim 2023, ss.103
Developing new transcriptional regulatory networks by modifying existing cis- and trans-acting modules is a key mechanism to design novel recombinant protein expression systems. Aca2, Mxr1, and Cat8 TFBSs are efficient tools to engineer the regulation and strength of Pichia pastoris AOX1 promoter. Engineered PmAOX1 exhibited 2.0-fold stronger expression in response to methanol induction, and the ethanol-repressed nature of PAOX1 was altered into the ethanol-inducible PmAOX1(Ergün et al., 2020).
Here, we focused on the impact of P. pastoris transcription factors Aca1, Mxr1, Cat8-1, and Cat8-2 on cell metabolism and transcriptional regulation. We created overexpression and knock-out strains to elucidate promoter regulation and establish innovative recombinant protein expression systems. Mxr1 overexpression enhanced PmAOX1 strength in response to ethanol and methanol while maintaining tight regulation on glucose and glycerol. Deletion of MXR1 resulted in the complete loss of PAOX1 activity.
Conversely, engineered PmAOX1 transcriptional strength was enhanced in the mxr1Δ mutant, suggesting that the promoter's dependency on Mxr1 was abolished through the integration of synthetic cis-acting elements. Aca1 acted as a methanol responsive transcriptional activator. P. pastoris cat8-1Δcat8-2Δ double knock-out mutant lost its ability to grow on ethanol. In the end, through rewiring transcriptional circuitry we developed novel protein expression profiles using ethanol, limited-glucose, glycerol, and methanol, and reached up to 5.6-fold stronger recombinant protein-production capacity.