Directed Metabolic Pathway Evolution Enables FunctionalPterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichiacoli
2020
Tetrahydrobiopterin-dependent
hydroxylation of aromatic amino acids
is the first step in the biosynthesis of many neuroactive compounds
in humans. A fundamental challenge in building these pathways in Escherichia coli is the provision of the non-native hydroxylase
cofactor, tetrahydrobiopterin. To solve this, we designed a genetic
selection that relies on the tyrosine synthesis activity of phenylalanine
hydroxylase. Using adaptive laboratory evolution, we demonstrate the
use of this selection to discover: (1) a minimum set of heterologous
enzymes and a host folE (T198I) mutation for achieving
this type of hydroxylation chemistry in whole cells, (2) functional
complementation of tetrahydrobiopterin by indigenous cofactors, and
(3) a tryptophan hydroxylase mutation for improving protein abundance.
Thus, the goal of having functional aromatic-amino-acid hydroxylation
in E. coli was achieved through directed metabolic
pathway evolution.
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