Research Projects

Recombinant protein production (RPP) in the methylotrophic yeast, P. pastoris, has been conventionally demonstrated to yield high protein titres by using a strong, methanol-inducible, and tightly regulated Alcohol Oxidase-1 promoter (pAOX1). However, during methanol induction phase in high-cell-density fermentation, the flammable nature and the accumulation of harmful reactive oxygen species (ROS) presents an unwanted bioprocess control challenge (Pan et al., 2022; De Brabander et al., 2023). Here, we propose to engineer a P. pastoris strain for methanol-free induction of RPP under pAOX1 for the development of safer and more efficient large-scale protein production process. The first objective involves CRISPR/Cas9-based genome editing or homologous recombination to engineer the methanol utilization pathway regulators (Fischer et al., 2022), wherein transcriptional repressor genes such as Nrg1 or Mig1 will be derepressed and transcriptional activator genes such as Mxr1 or Mit1 will be overexpressed under the promoter of a catabolite repressor such as pCAT1 (Chang et al., 2018; Vogl et al., 2018; Haghighi Poodeh et al., 2022). The pAOX1 driven heterologous gene in the engineered Pichia strain will be switched-on under the catabolite derepressed conditions, while retaining the powerful induction capacity of the pAOX1. As an alternative approach, a formate-based induction approach will also be explored by disrupting the FDH1 gene, enabling formate to serve as a stable and effective inducer of pAOX1-driven expression while using non-repressing carbon source like sorbitol (Singh & Narang, 2020; Liu et al., 2022; Feng et al., 2022). In the second objective, two of the industrially relevant enzymes, laccase and lipase, will be used as model proteins to assess the performance of engineered Pichia strain for its high-level expression capability under methanol-free conditions. The third objective focuses on using CRISPR/Cas9 genome editing strategy for the insertion of multiple copies of target genes downstream of the pAOX1 to boost expression levels without using selectable markers. (Gassler et al., 2019; Gao et al., 2022). Finally, bioprocess modeling will be used to characterize the engineered Pichia pastoris strain and optimize specific protein productivity (Qp) as a function of the specific growth rate (µ) for the development of continuous RPP process (Nieto-Taype et al., 2022; Jaswal et al., 2024). This model-based approach will uncover the optimal operational parameters associated with the feeding strategy to avoid the carbon-overflow metabolite formation during both the growth and the induction phase for industrial scalability and high volumetric productivity.