Figure 4. Application of biosensors to metabolic engineering in yeast.
(a) Fold activation of GL77F-PRO1-V by a panel of steroids in yEGFP reporter strain PyE1. Data are represented as mean ± SEM. (b) Growth of degron-G-PRO1-V in HIS3 reporter strain PJ69-4a is stimulated by progesterone but not pregnenolone. (c) Schematic for directed evolution of 3β-HSD using TF-biosensors for the conversion of pregnenolone to progesterone. (d) Fold activation of GL77F-PRO1-V by a panel of plasmids expressing wild-type 3β-HSD under varying promoter strengths in yEGFP reporter strain PyE1 when incubated in 50 μM pregnenolone. Data for plasmids containing CEN/ARS and 2 μ (2 micron) origins are shown. (e) Fold activation of GL77F-PRO1-V by a panel of evolved 3β-HSD mutants expressed under the TDH3 promoter on a CEN/ARS plasmid and incubated in 50 μM pregnenolone. (f) Progesterone titer in 1 OD of cells produced by strains expressing 3β-HSD mutants. Progesterone became toxic at levels of 100 μM and above, leading to substantial cell death. β-estradiol and hydrocortisone were not soluble in yeast growth media at levels above 25 μM. In a and d-f, data are presented as mean ± s.e.m. of three biological replicates. In d and e, (-) indicates cells lacking 3β-HSD. *indicates significance with a threshold of p < 0.05 using 2-tailed Student’s t-test.
Figure 4—figure supplement 1. Specificity of PRO biosensors enables selection for auxotrophy complementation.
