Uses of the Mtase selection system.

(A) ALE-driven workflow. (B) In vivo enzymatic comparison of wild-type Pnmt and variant shown in time course. n = 4, and error bars indicate SD. (C) The melatonin pathway. (D) In vivo enzymatic activity of Asmt and Aanat variants after 6 h cell growth. See S2 Fig for details. n = 3, and error bars indicate SD. (E) Comt-dependent growth shown using an evolved isolate bearing RpoC (A328P). μ indicates the growth rate. Inhibitor titration curves of the same strain in the presence or absence of homocysteine. n = 4, and error bars are SD. Underlying data can be found in S1 Data. Aanat, aralkylamine N-acetyltransferase; AcHT, acetylserotonin; AcCoA, acetyl-CoA; ALE, adaptive laboratory evolution; Asmt, acetylserotonine O-methyltransferase; Comt, catechol O-methyltransferase; Ddc, aromatic-amino-acid decarboxylase; Mtase, methyltransferase; NGS, next-generation sequencing; OCT, octopamine; OD, optical density; PCA, protocatechuic acid; Pnmt, phenylethanolamine N-methyltransferase; RpoC, RNA polymerase subunit beta; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SD, standard deviation; SYN, synephrine; VIII, vanillic acid; 5HT, serotonin; 5HTP, 5-hydroxytryptophan.