Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Pichia pastoris (Komagataella phaffii) is a key host for industrial heterologous protein production.
• Strain selection is a bottleneck due to clonal variability from random gene integration.
• Current screening methods are protein-specific and labor-intensive.

Purpose of the Study:

• To develop a generic, protein-based biosensor for high-throughput screening of Pichia pastoris transformants.
• To enable rapid identification of highly productive protein secretion clones.
• To overcome limitations of existing screening methodologies.

Main Methods:

• Engineered a Pichia pastoris strain with a split green fluorescent protein (GFP) biosensor.
• Fused the large GFP fragment (GFP1-10) to Tobacco Etch Virus (TEV) protease and targeted it to the endoplasmic reticulum.
• Tagged recombinant proteins with the small GFP fragment (GFP11) for secretion.
• Utilized GFP fluorescence as a direct readout for protein production levels.

Main Results:

• The split GFP biosensor system was successfully demonstrated for four recombinant proteins: phytase, laccase, β-casein, and β-lactoglobulin.
• Biosensor fluorescence levels directly correlated with traditional protein production assays.
• The system allows for secretion of untagged proteins of interest while retaining mature GFP intracellularly.

Conclusions:

• The developed split GFP biosensor provides a facile, generic, and rapid method for screening Pichia pastoris clones.
• This technology significantly improves the efficiency of identifying high-yield protein production strains.
• The biosensor system has broad applicability for recombinant protein production in Pichia pastoris.