Table 1.
The strain and plasmid used in this study.
Fig 1.
The effect of mutants in 5’-gene sequences on L-AAD catalyst activity, cell growth and KIC production.
a and b: The effect of mutant at 5’-gene sequences on biocatalyst activity and cell growth, respectively. c and d: The comparison of KIC production and bioconversion of BL21/A2 and (blue line) and BL21/pET28a-lad (green line). The arrows showed the additon of L-leucine.
Fig 2.
The effect of ribosome binding site design on L-AAD catalyst activity, cell growth and KIC production.
a: The comparison of biocatalyst activity with predicted translation rate. Gray bar: the biocatalyst activity and the BL21/pET28a-lad as the control (the yellow bar). ☆: predicted translation initiation rate. b: The effect of RBS mutants on cell growth. c and d: the KIC production and bioconversion rate of the 4 dominant RBS mutants and BL21/pET28a-lad. BL21/RBS3 (orange squares), BL21/RBS4 (green circles), BL21/RBS6 (black triangles), BL21/RBS8 (magenta triangles), BL21/pET28a-lad (violet rhombus), the additon of L-leucine (arrows).
Fig 3.
The effect of different origin on cell growth, biocatalyst activity, transcriptional level and KIC production.
a: the actual plasmid copy number under different origin. b: The effect of different plasmid origin on cell growth. c: The effect of different origin on biocatalyst activity. d: The qRT-PCR of L-AAD in different origin strains. e: The α-ketoisocaproate production of different origin strains f: The bioconversion rate of different origin strains. ColA (orange squares), p15A (blue triangles), CDF (green circles), pBR322 (red triangles), RSF (magenta triangles), pBR322* from BL21/pET28a-lad (black rhombus), the additon of L-leucine (arrows).
Fig 4.
The effect of integration of dominant conditions at transcription and translation.
a: the effect of integration of dominant conditions on biocatalyst activity. b: the effect of integration of dominant conditions on cell growth. c and d: The KIC production and bioconversion rate of integrated strains and BL21/pET28a-lad. BL21/ACYCDuet-RBS6 (blue squares), BL21/ACYCDuet-A2 (green circles), BL21/A2-RBS6 (red triangles), BL21/ACYC-A2-RBS6 (violet triangles), BL21/pET28a-lad (black rhombus), the additon of L-leucine (arrows).
Fig 5.
The reliability of tune translation level strategies and the SDS-PAGE analysis of protein expression.
a: For 5’-gene sequence mutants, the biocatalyst activity was compared with minimal folding free energy (ΔG) value. b: For RBS mutants, the biocatalyst activity was compared with the total free energy ΔGtot. c: SDS-PAGE analysis of total cell lysates of recombinant E. coli with different RBS sequences. d: SDS-PAGE analysis of cell lysate supernatant of recombinant E. coli with different RBS sequences. e: SDS-PAGE analysis of total cell lysates of recombinant E. coli at different plasmid copy number. f: SDS-PAGE analysis of cell lysate supernatant of recombinant E. coli at different plasmid copy number. Band indicated by arrow.