Convenient Genetic Encoding of Phenylalanine Derivatives through Their α-Keto Acid Precursors

The activity and function of proteins can be improved by incorporation of non-canonical amino acids (ncAAs). To avoid the tedious synthesis of a large number of chiral phenylalanine derivatives, we synthesized the corresponding phenylpyruvic acid precursors. Escherichia coli strain DH10B and strain C321.ΔA.expΔPBAD were selected as hosts for phenylpyruvic acid bioconversion and genetic code expansion using the MmPylRS/pyltRNACUA system. The concentrations of keto acids, PLP and amino donors were optimized in the process. Eight keto acids that can be biotransformed and their coupled genetic code expansions were identified. Finally, the genetic encoded ncAAs were tested for incorporation into fluorescent proteins with keto acids.

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Faculty Opinions recommendation of A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1057894.512855 ◽

2007 ◽

Author(s):

Niles Lehman

Keyword(s):

Genetic Code ◽

Genetic Encoding ◽

Genetic Code Expansion

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The influence of radiomimetic substances on deoxyribonucleic acid synthesis and function studied in escherichia coli/phage systems - IV. Colony-forming ability and escherichia coli strain B and two of its mutants following alkylation

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1959.0055 ◽

1959 ◽

Vol 151 (942) ◽

pp. 129-147 ◽

Cited By ~ 4

Keyword(s):

Escherichia Coli ◽

Chemical Reactivity ◽

Alkylating Agents ◽

Chemical Change ◽

Acid Synthesis ◽

Coli Strain ◽

Ribonucleic Acids ◽

Significant Difference ◽

Synthetic Capacity ◽

And Function

Escherichia coli strain B , its mutant B/r and a new mutant, designated B/HN 2, have been employed in a study of the effect of alkylating agents upon the survival of colony-forming ability and phage-synthetic capacity. This has been done against the background of our earlier work upon phage and that of other workers upon bacteria, employing both alkylating agents and radiations. The sensitivity of B toward all the compounds now studied was greater than that of the other two strains as regards colony-forming ability, whereas all three strains showed a similar sensitivity in regard to capacity. Survival curves of all strains treated with monofunctional agents were of a so-called ‘multi-hit’ type, whereas those for bifunctional compounds were downwardly concave. The response to di(2-ehloroethyl) methylamine ( HN 2) was complicated by the chemical change undergone by this substance in aqueous solution, as was shown by a comparison of fresh and aged solutions and of the effect of different cultural conditions prior to treatment. As with radiations, phage-synthetic capacity was considerably less sensitive to alkylation than colony-forming ability, whilst this sensitivity was essentially the same for the three strains. No significant difference was found between the effect of HN 2 on the capacity of B for T 2 and for T 7. The capacity of B/r for T 2 was more sensitive to treatment by a bifunctional agent than by a monofunctional agent of similar chemical reactivity. It is suggested that this may implicate ribonucleic acids as the reactive substrate essential to capacity.

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Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Nucleic Acids Research ◽

10.1093/nar/gkq080 ◽

2010 ◽

Vol 38 (11) ◽

pp. 3682-3691 ◽

Cited By ~ 25

Author(s):

Fumie Iraha ◽

Kenji Oki ◽

Takatsugu Kobayashi ◽

Satoshi Ohno ◽

Takashi Yokogawa ◽

...

Keyword(s):

Escherichia Coli ◽

Genetic Code ◽

Trna Synthetase ◽

Genetic Code Expansion ◽

Functional Replacement

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A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0610294104 ◽

2007 ◽

Vol 104 (3) ◽

pp. 1021-1026 ◽

Cited By ~ 66

Author(s):

David G. Longstaff ◽

Ross C. Larue ◽

Joseph E. Faust ◽

Anirban Mahapatra ◽

Liwen Zhang ◽

...

Keyword(s):

Genetic Code ◽

Genetic Encoding ◽

Genetic Code Expansion

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Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

Frontiers in Microbiology ◽

10.3389/fmicb.2021.744284 ◽

2021 ◽

Vol 12 ◽

Author(s):

Huawei Yi ◽

Jing Zhang ◽

Famin Ke ◽

Xiurong Guo ◽

Jian Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Expression ◽

Rna Polymerase ◽

T7 Rna Polymerase ◽

Noncanonical Amino Acids ◽

E Coli ◽

Growth Defects ◽

Genetic Code Expansion ◽

And Function

Incorporation of noncanonical amino acids (ncAAs) into proteins has been proven to be a powerful tool to manipulate protein structure and function, and to investigate many biological processes. Improving the yields of ncAA-containing proteins is of great significance in industrial-scale applications. Escherichia coli C321.ΔA was generated by the replacement of all known amber codons and the deletion of RF1 in the genome and has been proven to be an ideal host for ncAA-containing protein expression using genetic code expansion. In this study, we investigated the transcriptome and proteome profiles of this first codon reassignment strain and found that some functions and metabolic pathways were differentially expressed when compared with those of its parent strain. Genes involved in carbohydrate and energy metabolism were remarkably downregulated. Our results may provide important clues about the growth defects in E. coli C321.ΔA. Furthermore, we improved the yields of ncAA-containing proteins in E. coli C321.ΔA by integrating the T7 RNA polymerase system.

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Phosphoregulation of tropomyosin-actin interaction revealed using a genetic code expansion strategy

Wellcome Open Research ◽

10.12688/wellcomeopenres.16082.1 ◽

2020 ◽

Vol 5 ◽

pp. 161

Author(s):

Saravanan Palani ◽

Darius Koester ◽

Mohan K. Balasubramanian

Keyword(s):

Escherichia Coli ◽

Genetic Code ◽

Actin Filaments ◽

Total Internal Reflection ◽

Coiled Coil ◽

Trna Synthetase ◽

Expansion Strategy ◽

Direct Binding ◽

Genetic Code Expansion ◽

The Stability

Tropomyosins are coiled-coil proteins that regulate the stability and / or function of actin cytoskeleton in muscle and non-muscle cells through direct binding of actin filaments. Recently, using the fission yeast, we discovered a new mechanism by which phosphorylation of serine 125 of tropomyosin (Cdc8), reduced its affinity for actin filaments thereby providing access for the actin severing protein Adf1/Cofilin to actin filaments causing instability of actin filaments. Here we use a genetic code expansion strategy to directly examine this conclusion. We produced in Escherichia coli Cdc8-tropomyosin bearing a phosphate group on Serine-125 (Cdc8PS125), using an orthogonal tRNA-tRNA synthetase pair that directly incorporates phosphoserine into proteins in response to a UAG codon in the corresponding mRNA. We show using total internal reflection (TIRF) microscopy that, whereas E.coli produced Cdc8PS125 does not bind actin filaments, Cdc8PS125 incubated with lambda phosphatase binds actin filaments. This work directly demonstrates that a phosphate moiety present on serine 125 leads to decreased affinity of Cdc8-tropomyosin for actin filaments. We also extend the work to demonstrate the usefulness of the genetic code expansion approach in imaging actin cytoskeletal components.

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