Reliable high-throughput approach for screening of engineered constitutive promoters in the yeast Pichia pastoris

Aims: To develop a reliable and sensitive high-throughput approach for the screening of engineered constitutive promoters in the yeast Pichia pastoris.

Methods and results: The yeast-enhanced green fluorescent protein (yEGFP) was used as the reporter to monitor the promoter strength. After eliminating the interfering components (yeast extract and tryptone) with fluorescence signal from the medium, a high-throughput screening approach was established and optimized to obtain a low standard deviation of cell density (6.9%) and fluorescence (7.4%) in 48-deep-well microplates. Then, 300 clones containing GAP promoter (P(GAP)) variants were screened, exhibiting a wide range in fluorescent intensity from about 8% to 218% of that obtained with P(GAP). Six representative clones with unique promoter sequence were picked for further characterization. A good correlation between yEGFP fluorescence in microplates and shake flasks was observed. Furthermore, the high correlation between fluorescence and transcript levels confirmed that expression was transcriptionally controlled.

Conclusions: We developed a reliable high-throughput screening approach that can be used to select engineered constitutive promoters of varying strengths.

Significance and impact of the study: This approach is expected to accelerate the selection of constitutive promoters in P. pastoris and can also be applied for the screening of other constitutive expression clones.