Fig. 4: Construction and screening of tailored promoter libraries for target genes.
a Workflow of construction and screening of tailored promoter libraries. The reporter gene was constructed by fusing the first 180 bp of the target gene (gdh, pyc, or proB) with a flexible linker (GGGGS)3 and an rfp gene. Random mutation libraries of native promoters were constructed by PCR with primers containing several degenerate bases. The plasmid libraries (~106) were transformed into C. glutamicum. Approximately 1/10 transformants were first screened by colony fluorescence imaging and those with increased RFP fluorescence were cultivated in 96-deep-well plates for a second round of screening. b Representative gdh and pyc promoter libraries tested in this study. The sequences of wild-type promoter and random mutation library are shown. The degenerate bases introduced during PCR primer synthesis are highlighted in red. The predicted -35 and -10 regions are highlighted in black boxes. Other tested promoter libraries for gdh, pyc, and proB are shown in Supplementary Fig. 7. c Strength analysis of the Pgdh variants using the gdh-rfp fusion gene as a reporter. WT represents the wild-type Pgdh control. The number represents the serial number of Pgdh variants. Data are presented as mean values +/− SD (n = 3 independent experiments). d Strength analysis of the Ppyc variants using the pyc-rfp fusion gene as a reporter. WT represents the wild-type Ppyc control. The number represents the serial number of Ppyc variants. Data are presented as mean values +/− SD (n = 3 independent experiments). e Strength analysis of Pgdh-29 and Ppyc-20 using the proB-rfp fusion gene as a reporter. All the PproB libraries failed to generate good diversity and the strongest Pgdh-29 and Ppyc-20 variants were used for expression of the proB-rfp fusion gene. The wild-type PproB was used as a control. Data are presented as mean values +/− SD (n = 3 independent experiments). Cells of the stationary growth phase were used to detect their fluorescence outputs using a microplate reader (λ excitation = 560 nm, λ emission = 607 nm). Source data underlying Fig. 4c–e are provided as a Source Data file.
