Results: 3

1.
Figure 2

Figure 2. From: A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation.

Chemical complementation detects bond cleavage reactions A. In the cellulase growth selection, the Methotrexate-Cellotetraose-Dexamethasone (Mtx-Cel-Dex) heterodimer links a DNA-binding domain fused to dihydrofolate reductase (DBD-DHFR) and an activation domain fused to the glucocorticoid receptor (AD-GR), activating transcription of the toxic URA3 reporter gene, thus leading to cell death. Active cellulases cleave the β-1,4-glucosidic bond in the Mtx-Cel-Dex linker, decreasing transcription of the toxic URA3 reporter gene, and leading to cell survival. B. Structure of the Mtx-Cel-Dex chemical inducer of dimerization used for this assay. C. The URA3 counter selection strain was constructed in three steps. First, the URA3 gene was introduced at the ura3-52 locus of the chemical complementation yeast strain (FY251) via homologous recombination. Next, the AD-GR and the DBD-DHFR constructs were introduced into FY251 via transformation. Finally, the LexA DNA binding site and the tightly regulated Spo13 promoter were introduced upstream of the URA3 reporter gene via homologous recombination.

Pamela Peralta-Yahya, et al. J Am Chem Soc. ;130(51):17446-17452.
2.
Figure 1

Figure 1. From: A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation.

Chemical complementation provides a high-throughput selection for cellulase catalysts. The discovery of improved cellulases, and other hydrolase enzymes important for biomass conversion is limited by existing medium-throughput screening technologies. Chemical complementation has been adapted to provide a high-throughput selection for bond cleavage reactions and specifically cellulose catalysts. Chemical complementation detects enzyme catalysis of bond formation or cleavage reactions based on covalent coupling of two small molecule ligands. The heterodimeric small molecule reconstitutes a transcriptional activator, turning on the transcription of a downstream reporter gene. Here, a dexamethasone (Dex)-methotrexate (Mtx) yeast three-hybrid system is used. Endoglucanase activity is detected as cleavage of a glycosidic linkage between Mtx and Dex. A cellulase selection could be used as an initial high-throughput assay to evaluate very large libraries of cellulase variants generated either by mutagenesis or isolated from biodiversity sources. Active cellulases emerging from the high-throughput assay could be then tested using low-throughput assays using plant material that more closely mimic industrial conditions.

Pamela Peralta-Yahya, et al. J Am Chem Soc. ;130(51):17446-17452.
3.
Figure 3

Figure 3. From: A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation.

The chemical complementation URA3 counter selection strain. A. The URA3 reverse yeast three-hybrid system. Absence of small molecule leads to cell survival and an increase in absorption at 600nm. Presence of either 1, 5, 10 µM Mtx-Dex, or 1 µM Mtx-Cel-Dex leads to toxic URA3 reporter gene transcription, cell death, and a decrease of absorption at 600nm. The experiments were carried out in triplicate. Shown is the mean growth and the error bars represent the standard deviation from the mean. B. Reversion rate of the chemical complementation URA3 counter selection strain. Ninety three unique chemical complementation URA3 counter selection conditions (1μ M MTX-Dex) and under non-counter selection conditions (No MTX-Dex) as a control. Shown is the mean growth and the error bars represent the standard error form the mean (p-value<0.0001). C. The chemical complementation URA3 counter selection strain detects cellulase activity. Presence of 1 µM Mtx-Cel-Dex and active cellulase Cel7B leads to cell survival and an increase in absorption at 600 nm. Presence of 1 µM Mtx-Cel-Dex and inactive cellulose Cel7B:E197A leads to cell death and a decrease in absorption at 600nm. As a control, the URA3 counter selection strain was also grown in the absence of Mtx-Cel-Dex and either the presence of Cel7B or Cel7B:E197A. All experiments were carried out in triplicate. Shown is the mean growth and the error bars represent the standard deviation from the mean. D. The chemical complementation URA3 counter selection enriches for cellulase activity after five days of selection. Twenty-two cellulase variants isolated before selection and twenty-two variants isolated after five days of selection were tested for cellulase activity using carboxymethylcellulose (CMC) in crude cell extracts. Increase in aldehyde formation was detected as an increase in absorption at 660nm. Shown is the mean cellulase activity and the error bars represent the standard error form the mean (p-value<0.005).

Pamela Peralta-Yahya, et al. J Am Chem Soc. ;130(51):17446-17452.

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