FIG 1.

Route for the directed evolution of UPO1 toward functional expression and improved activity. New mutations are depicted as stars and accumulated mutations as squares. Mutations in the mature PaDa-I mutant and their origins are boxed. The signal peptide is represented in dark red and the mature protein in red. In the parental n-UPO1, the glycosylation sites (Asn11, Asn141, Asn161, Asn182, Asn286, and Asn295) are represented as blue stars, the Phe triad (Phe69, Phe121, and Phe199) involved in the binding of aromatic substrates is marked with green arrows, and the acid-base pair for peroxide cleavage (Glu196 and Arg189) is indicated with blue arrows. TAI represents the improvement in UPO1 activity detected in S. cerevisiae microcultures for each mutant compared with the parental n-UPO1. Thermostability (T₅₀) was estimated from culture supernatants (Fig. 2B). The breakdown of the TAI into specific activity and expression is shown in Fig. 3. The dashed arrows indicate the parental types used for each round of evolution. In the 3rd generation, “a” indicates the offspring obtained by IvAM of 12C12 and “b” indicates the offspring obtained by MORPHING in the signal peptide of 12C12. In the 4th generation, “c” indicates the triple mutant at the signal peptide constructed using I13D3 as a template and “d” the offspring obtained by mutagenic PCR and shuffling of parents I13D3, M5D2, and M4D8. n.d., not determined; n.m., not measurable. (See also Table S1 in the supplemental material.)