Fig. 6

Synthetic glycine-OFF riboswitch used for the directed evolution of alanine-glyoxylate aminotransferase in E. coli. a A mutant of AGXT with higher enzyme activity was screened using a high-throughput screening platform based on the synthetic glycine-OFF riboswitch. The genes agxtM, aceA and hemA were co-expressed from two plasmids to syntheze 5-ALA in E. coli. The glyoxylate pathway is highlighted in red. b The library of agxT mutants was constructed using error-prone PCR (Additional file 1: Table S6). The library plasmids and puc18-GlyOFF6-aceA plasmid were co-expressed in E. coli MG1655. c All clones from LB plates were transferred to M9 medium, and selected in fresh M9 medium supplemented with 100 μg mL⁻¹ ampicillin and 90 μM Ni²⁺ to remove the strains with lower levels of AGXT enzyme activity. AGXT mutants with higher activity were enriched in three cycles, and the plasmids from each cycle were extracted and transferred to the original strain. The percentage of beneficial mutations and enzyme activity were also determined. The relevant genes include aceA, encoding isocitrate lyase; agxTM, encoding mutant glyoxylate transaminase; hemA, encoding 5-aminolevulinic acid synthase; tetA, tetracycline resistance gene; gfp, encoding green fluorescent protein. Ac-CoA, acetyl coenzyme A; CIT, citrate; ICIT, isocitrate; GOX, glyoxylate; AKG, α-ketoglutaric acid; SUCCoA, succinyl-CoA; SUC, succinic acid; FUM, fumaric acid; MAL, malic acid; OAA, oxaloacetic acid; Gly, glycine; 5-ALA, 5-aminolevulinic acid; Amp: ampicillin; Cr: chloramphenicol; ori, replicon. d Specific enzyme activity of wild-type AGXT, three library strains, AGXT22, and AGXT26. e 5-aminolevulinic acid concentrations of MG1655K, MG1655-HAA, MG1655-HAA22, and MG1655-HAA26. All the data are the average values of three independent experiments.**P < 0.01