Fig. 3
From: Sustainable and high-level microbial production of plant hemoglobin in Corynebacterium glutamicum
Engineering of native promoters for inducible hemoglobin expression. A Catabolic pathway of propionate and gluconate in C. glutamicum. The red crosses represent gene knock-out to block the degradation of propionate and gluconate. B Comparison of different inducible promoters for hemoglobin expression. WT, the wild-type C. glutamicum strain. ∆prpDBC2, the C. glutamicum mutant with propionate catabolic genes prpDBC2 deleted. ∆gntK, the C. glutamicum mutant with gluconate catabolic gene gntK deleted. Control represents the wild-type strain transformed with an empty plasmid. The pXMJ19-Lba-Ec-gfp and the pXMJ19-mHb-Cg-gfp plasmids for IPTG-inducible expression of the hemoglobin–GFP fusions were transformed into the wild-type strain. The pXMJ19-PprpD2-Lba-Ec-gfp and the pXMJ19-PprpD2-mHb-Cg-gfp plasmids for propionate-inducible expression of the hemoglobin–GFP fusions were transformed into the wild-type strain and the ∆prpDBC2 strain. The pXMJ19-PgntK-Lba-Ec-gfp and the pXMJ19-PgntK-mHb-Cg-gfp plasmids for gluconate-inducible expression of the hemoglobin–GFP fusions were transformed into the wild-type strain and the ∆gntK strain. C Schematic diagram of the designed PprpD2 promoter libraries. The introduced degenerate codons are highlighted in red. D Characterization of selected PprpD2 promoter variants for hemoglobin expression. WT represents the wild-type PprpD2 promoter and the rest are promoter variants selected from the promoter libraries. 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. E Analysis of the sequence characteristics of the strong PprpD2 promoter variants selected from the random mutation library. 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)