Directed Metabolic Pathway Evolution Enables Functional Pterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichia coli

Abstract

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.

Microbial Synthesis of Human-Hormone Melatonin at Gram Scales

“…Heterologous TrpH was a truncated form of human TrpH2 with an E2K mutation for improved protein abundance. 2 Expression of the recombinant genes were individually achieved from a trc promoter except for pcd, whose expression was under the control of the E. coli aroF promoter. The background E. coli strain was a plasmidless isolate described in Luo et al (2020) 2 and it carried a chromosomal FolE (T198I) mutation that was essential for achieving efficient tryptophan hydroxylation.…”

“…2 Expression of the recombinant genes were individually achieved from a trc promoter except for pcd, whose expression was under the control of the E. coli aroF promoter. The background E. coli strain was a plasmidless isolate described in Luo et al (2020) 2 and it carried a chromosomal FolE (T198I) mutation that was essential for achieving efficient tryptophan hydroxylation. Deletion of tnaA (tryptophanase) and trpR (tryptophan transcription repressor) genes was additionally introduced.…”

“…9,10 Notably, the E. coli strain used was ΔtyrA, implemented as a part of the genetic selection for improving tryptophan hydroxylation. 2 Deletion of tyrA resulted in tyrosine auxotrophy. Although de novo tyrosine synthesis from phenylalanine via TrpH could be achieved in HM175, it might be limited under studied conditions thus representing a potential contributing factor for acetate formation during fermentation.…”

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

“…used a minimum set of heterologous enzymes and a host folE (T198I) mutation for achieving pterin-dependent l -tryptophan hydroxylation by directed metabolic pathway evolution. More important, a heterologously introduced BH4 biosynthetic pathway was deleted during adaptive laboratory evolution, the host strain repurposed one of its native cofactors, but it remained unclear exactly which E. coli pterin was repurposed as an alternative cofactor [ 66 ]. Heterologous expression of enzymes and the enzyme mutation folE (T198I) in E. coli can be used to increase the production of GTP and BH4.…”

“…A mutagenized TPH was expressed in E. coli , and 0.8 mM 5-HTP was synthesized after adding BH4 as a substrate. In addition, the BH4 regeneration pathway and glucose dehydrogenase of Bacillus subtilis were introduced to increase the utilization rate of BH4, and the production of 5-HTP was increased to 2.5 mM [ 38 , 47 , 66 ].…”