Fig. 3: Codon saturation mutagenesis of γ-glutamyl kinase of C. glutamicum (CgProB) for improved l-proline production via CRISPR/Cas9-assisted ssDNA recombineering. | Nature Communications

Fig. 3: Codon saturation mutagenesis of γ-glutamyl kinase of C. glutamicum (CgProB) for improved l-proline production via CRISPR/Cas9-assisted ssDNA recombineering.

From: CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction

Fig. 3

a Conservation analysis of γ-glutamyl kinases. The conserved region involved in l-proline binding is indicated in yellow. The A146, T147, T148, V150, and N151 residues mutated in this study are highlighted in green. The two gRNAs used and their corresponding PAM sequences (underlined) are highlighted in blue and purple, respectively. γ-Glutamyl kinases sequences used for conservation analysis are provided in Supplementary Data 1. b The model structure of CgProB. The model structure was constructed with the crystal structure of γ-glutamyl kinase from E. coli (PDB ID: 2J5T)80 as a template (93% coverage and 38% sequence identity with CgProB) using Discovery Studio 2018 software. The backbone of CgProB is shown in a ribbon model on which several key residues are shown in a stick model. The substrate l-glutamate is indicated in magenta. The conserved amino acid residues G153, D154, N155, and D156 are indicated in yellow. The engineered amino acids residues A146, T147, T148, V150, and N151 are indicated in green. The full structure is shown in Supplementary Fig. 5. c Growth and l-proline production by C. glutamicum strains with amino acid substitution at codon 146 of CgProB. The strain harboring reported CgProBG149K mutation35 was used as a control. l-Proline production was conducted in 96-deep-well plates. Data are presented as mean values +/− SD (n = 3 independent experiments). d, Growth and l-proline production by C. glutamicum strains with amino acid substitution at codon 150 of CgProB. The strain harboring reported CgProBG149K mutation35 was used as a control. The V150N mutation used in strain PRO-1 is indicated in red. l-Proline production was conducted in 96-deep-well plates. Data are presented as mean values +/− SD (n = 3 independent experiments). **P = 0.0024, Student’s two-tailed t-test. Source data underlying Fig. 3c, d are provided as a Source Data file.

Back to article page