Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli

Chen, Yuda; Loredo, Axel; Chung, Anna; Zhang, Mengxi; Li, Rui; Xiao, Han

doi:10.1016/j.jmb.2021.167412

Cited by 8 publications

(6 citation statements)

References 66 publications

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“…Additionally, a completely autonomous bacterial strain utilizing DOPA as a 21st amino acid for protein synthesis has been constructed. 22 The yield of DOPA-containing protein from this autonomous strain is greater than that from cells exogenously fed with 9 mM DOPA. More recently, an autonomous bacterium engineered for autonomous 5-hydroxytryptophan biosynthesis and incorporation has been used to monitor oxidative stress in real-time.…”

mentioning

confidence: 82%

“…Compared to exogenous feeding of 1 mM pThr, the biosynthetic system produced a 40‐fold higher level of intracellular pThr, resulting in greater incorporation efficiency. Additionally, a completely autonomous bacterial strain utilizing DOPA as a 21st amino acid for protein synthesis has been constructed 22 . The yield of DOPA‐containing protein from this autonomous strain is greater than that from cells exogenously fed with 9 mM DOPA.…”

Section: Introductionmentioning

confidence: 99%

“…In a limited number of examples, the employment of biosynthesized ncAAs has been shown to be beneficial for both the efficiency of ncAA incorporation and the application of genetic code expansion to biological systems. Several synthetic gene clusters for ncAA biosynthesis, including p-aminophenylalanine, 18,19 phosphothreonine (pThr), 20 5-hydroxytryptophan, 21 and 3,4-dihydroxyphenylalanine (DOPA), 22 have been coupled to their corresponding orthogonal mutant aaRS/tRNA pairs to generate completely autonomous E. coli cells with an expanded genetic code. In other examples, aminotransferase, tyrosine phenol-lyase, and cysteine β-replacement enzyme have been used to create E. coli strains able to biosynthesize and incorporate phenylalanine analogs, 23 DOPA, 24 and cysteine analogs 25 into proteins.…”

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confidence: 99%

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Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid

Moore

Miller

et al. 2022

Protein Science

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Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of Omethyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.

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confidence: 82%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid

Moore

Miller

et al. 2022

Protein Science

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“…Start‐of‐art genome editing technology also provides more options to conveniently generate engineered organisms containing stop codons in open reading frames through base‐editing technology [61] or the CRISPR‐Cas9 system [43] . The production of phosphor‐threonine, [17c] 5‐hydroxytryptophan, [62] DOPA [63] and sulfotyrosine [64] by coupling biosynthesis pathways with genetic coding has confirmed the feasibility of this approach, which does not require the addition of chemically synthesized CTM amino acids. It would be highly desirable to obtain a method to biosynthesize acylated lysine analogues in eukaryotes, in particular acetyllysine, which would bring the first‐step in the breakthrough to explore the biological or evolutional consequences of PTM versus CTM.…”

Section: Future Directionsmentioning

confidence: 99%

Studying Reversible Protein Post‐translational Modification through Co‐translational Modification

Liu

2023

ChemBioChem

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Understanding the post-translational modifications of targeted proteins is of great significance for manipulating the physiological processes of eukaryotes. Chemical biology tools have been used to investigate the biological roles of those posttranslational modifications at particular sites, especially genetic code expansion technology, which can also be combined with the concept of synthetic biology to generate a genetically modified organism with a synthetic auxotroph for co-translational modification components. In this concept, we will introduce applications, limitations, and perspectives of genetic code expansion technology for studying post-translational modification based on recent progresses. Future perspectives of genetically modified organisms also will be discussed in regard to the application of post-translational modification research.

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“…14 A similar strategy was recently applied to the creation of autonomous bacterial cells that can biosynthesize and genetically incorporate p -amino-phenylalanine ( p AF), 5-hydroxyl-tryptophan (5HTP) and dihydroxyphenylalanine (DOPA), although no autonomous eukaryotic cells have been reported. 17,19,20 We see there that additional biosynthetic pathways for producing polar or negatively-charged ncAAs would greatly expand the utility of genetic code expansion methods.…”

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confidence: 99%

Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

Chen

Jin

Zhang

et al. 2022

Preprint

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Incorporation of noncanonical amino acids (ncAAs) into proteins holds great promise for modulating the structure and function of those proteins and for influencing evolutionary dynamics in organisms. Despite significant progress in improving the efficiency of translational machinery needed for incorporating ncAAs, exogenous feeding of high concentrations of chemically-synthesized ncAAs, especially in the case of polar ncAAs, is required to ensure adequate intracellular ncAA levels. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. We discovered the first enzyme catalyzing tyrosine sulfation, sulfotransferase 1C1 from Nipponia nippon (NnSULT1C1), using a sequence similarity network (SSN). The unique specificity of NnSULT1C1 for tyrosine has been systematically explored using both bioinformatics and computational methods. This NnSULT1C1 was introduced into both bacterial and mammalian cells so as to yield organisms capable of biosynthesizing high levels of intracellular sTyr. These engineered cells produced site-specifically sulfated proteins at a higher yield than cells fed exogenously even with the highest level of sTyr reported in literature. We have used these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

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Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli

Cited by 8 publications

References 66 publications

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid

Studying Reversible Protein Post‐translational Modification through Co‐translational Modification

Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

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