Skip to main content
Figure 3 | BMC Systems Biology

Figure 3

From: A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

Figure 3

Optimization for stationary phase results in CatA variants that are improved at late growth. Ten CatA variants were generated, including wild-type and a version optimized by Blue Heron Biotechnology. The three stationary phase variants (S1-S3) were made from a codon usage matrix constructed using the 50 most highly expressed genes after three days of growth (see Supplementary Matrices). The two high-expression variants (H1-H2) were made from a codon usage matrix constructed using the 100 most highly expressed genes in yeast grown in rich media. The three control variants (C1-C3) were constructed from the control matrix). Two assays were conducted to measure activity. a. Cells expressing the CatA variants were grown for 6, 18 or 24 hours prior to bulk protein extraction. The Vmax (mM/min*μg protein) for conversion of catechol to muconic acid was determined for the bulk protein. Biological triplicates and technical triplicates were measured to determine standard deviations. b. Cells expressing the CatA variants were grown for 18 hours in 30 mL before spiking the media with 1 g/L of catechol. After 24 additional hours of growth, 1 mL of supernatant was extracted and analyzed using HPLC, as previously described [22], to determine total muconic acid production. Normalized muconic acid levels (mg/L*OD600) are reported and standard deviation was determined using biological triplicates and p-values were calculated using a t-test to determine statistical significance (described in text of paper).

Back to article page