Fig. 2

Optimization of the N-terminal coding sequence of hemoglobin. A Schematic diagram of saturated synonymous mutation for the first 12 codons of Lba-Ec and mHb-Cg. The pie charts represent the codon usage frequency in C. glutamicum. B Schematic diagram of NCS library construction and screening using the fluorescence imaging system. C Representative image of the screening agar plates got by the fluorescence imaging system. Colonies with relatively high fluorescence intensities are marked with red arrows. D Second round of screening by cultivation in 96-well plates. E Characterization of the selected NCS variants from the Lba-Ec library by cultivation in shaking flasks. To remove the possible mutations in the plasmid backbone or the chromosome that were randomly introduced during library construction and screening, the expressing plasmids were reconstructed based on the sequencing data and transformed into C. glutamicum for test. F Characterization of the selected NCS variants from the mHb-Cg library by cultivation in shaking flasks. To remove the possible mutations in the plasmid backbone or the chromosome that were randomly introduced during library construction and screening, the expressing plasmids were reconstructed based on the sequencing data and transformed into C. glutamicum for test. G Codon relative adaptiveness of the first 12 codons of the selected variants with the codon usage frequency of C. glutamicum genome as a reference. The hemoglobin expression levels were characterized by measuring the fluorescence intensities of hemoglobin–GFP fusion. Values and error bars represent means and standard deviations (n = 3)