Fig. 2: Non-canonical amino acid (ncAA) incorporation tool-based pathway regulation in genetically recoded Escherichia coli Δ321AM.
a Testing the efficiency of ncAA tool-pEVOL using the reporter protein GFP (left panel). The histogram (middle panel) represents the degree of GFP expression recovery after replacing one or two original codons by amber stop codons in the sequence from the second to the seventh codon (2TAG to 7TAG and 23TAG to 67TAG). The data are expressed as the mean ± SD from six (n = 6) biologically independent replicates. The line graph (right panel) shows the use of the 2TAG GFP gene to test GFP expression levels in the presence of different concentrations of p-acetyl-L-phenylalanine (pAcF). The data are expressed as the mean ± SD from three (n = 3) biologically independent replicates. b Intracellular metabolic feedback loop of engineered E. coli. ‘T’ marker represents the regulation of metabolic flow. c Construction of the N-acetylglucosamine (GlcNAc) biosynthetic E. coli Δ321AM strain. Red circular symbols represent gene knockouts, the ‘T’ marker represents the regulation of metabolic flux, and bold arrows represent an increase in metabolic flux. d Model simulated (half-saturation constant KS = 15) and actual fermentation results of the three GlcNAc biosynthetic strains measured in the presence of different pAcF concentrations. The data are expressed as the mean ± SD from three (n = 3) biologically independent replicates. Source data underlying (a), (d) are provided as a Source data file.
