将生物正交化学与人工代谢相偶联：叠氮高丙氨酸的细胞内生物合成及其在重组蛋白中的掺入。
Coupling bioorthogonal chemistries with artificial metabolism: intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins.
机构信息
Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany.
出版信息
Molecules. 2014 Jan 15;19(1):1004-22. doi: 10.3390/molecules19011004.
In this paper, we present a novel, "single experiment" methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of L-azidohomoalanine from O-acetyl-L-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph E. coli strain. In our system, the host's methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient L-azidohomoalanine incorporation in place of L-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.
在本文中，我们提出了一种新颖的“单实验”方法，该方法基于代谢途径的遗传工程，用于从简单的前体物直接在细胞内产生非典型氨基酸，并扩展遗传密码。具体来说，我们通过将大肠杆菌的 AUG 密码子重新分配，将 L-叠氮高丙氨酸从 O-乙酰-L-同型丝氨酸和 NaN3 工程化到细胞内生物合成中，并在甲硫氨酸营养缺陷型大肠杆菌菌株中实现其直接掺入重组靶蛋白。在我们的系统中，首先通过利用来自谷氨酸棒杆菌的重组吡哆醛磷酸依赖型 O-乙酰同型丝氨酸硫基转移酶的广泛反应特异性，将宿主的蛋氨酸生物合成途径转向产生所需的非典型氨基酸。然后，表达靶蛋白 barstar，并有效地用 L-叠氮高丙氨酸取代 L-蛋氨酸进行掺入。这项工作证明了这一原理，并为从常见的可发酵来源直接在细胞内生产和定点掺入生物技术相关的非典型氨基酸的进一步工作铺平了道路。
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