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.