Fig. 3

RAPID screening for the improvement of tyrosine production through an evolved Aro4p. The RAPID screening approach was used to identify novel mutations in aro4 that improve tyrosine production by yeast. Libraries were constructed through mutagenizing the proposed regulatory region of Aro4p (residues 191–263) a–c and through whole-gene mutagenesis of the best previously reported variant aro4-K229L d–f. Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process (a, d). Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for tyrosine production with the sorted clones having a 1.9- and 5.8-fold increase in secretion compared to the unsorted clones for Aro4p and K229L clones, respectively (b, e). Crystal structures of Aro4p are derived from PDB 1OF6 and marked with the identified mutations (c, f). Error bars represent standard error of biological triplicates. Tyrosine production was increased nearly 28-fold over the wild-type production using this approach