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

Abstract

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.

Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids

“…Next, we tested the amber suppression activity of MVTyrRS to explore its performance in the RF1 knockout strain. The above plasmid transformations were repeated in E. coli C321.∆A.exp∆PBAD [ 36 ]. The results indicated that, in the C321.∆A.exp∆PBAD strain, the fluorescence intensity of the MVTyrRS was similar to that in the DH10B strain, as was the MjTyrRS ( Figure 3 C).…”

“…The E. coli DH10B strain was purchased from Tsingke Biotechnology. The E. coli C321.∆A.exp∆PBAD strain [ 36 ] was previously constructed in our lab based on E. coli C321.∆A.exp.…”