Directed Metabolic Pathway Evolution Enables Functional Pterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichia coli.,ACS Synthetic Biology

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Directed Metabolic Pathway Evolution Enables Functional Pterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichia coli.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-03-09 , DOI: 10.1021/acssynbio.9b00488
Hao Luo ₁ , Lei Yang ₁ , Se Hyeuk Kim ₁ , Tune Wulff ₁ , Adam M Feist _{1,

2} , Markus Herrgard ₁ , Bernhard Ø Palsson _{1,

2,

3}

Affiliation

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.

中文翻译：

定向代谢途径进化使大肠杆菌中功能性蝶呤依赖性芳香氨基酸羟基化成为可能。

芳香族氨基酸的四氢生物蝶呤依赖性羟基化是人类许多神经活性化合物生物合成的第一步。在大肠杆菌中构建这些途径的一个基本挑战是提供非天然羟化酶辅因子四氢生物蝶呤。为了解决这个问题，我们设计了一种依赖于苯丙氨酸羟化酶的酪氨酸合成活性的遗传选择。利用适应性实验室进化，我们证明了利用这种选择来发现：(1) 一组最小的异源酶和宿主 folE (T198I) 突变，用于在全细胞中实现这种类型的羟基化化学，(2) 四氢生物蝶呤的功能互补通过本土辅因子，以及（3）色氨酸羟化酶突变来提高蛋白质丰度。因此，通过定向代谢途径进化实现了在大肠杆菌中实现功能性芳香族氨基酸羟基化的目标。

更新日期：2020-03-09

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