Construction and characterization of a mutant library for the P₂₃ constitutive promoter in lactic acid bacteria

Promoters are crucial elements for controlling gene expression in cells, yet lactic acid bacteria (LAB) often lack a diverse set of available constitutive promoters with quantitative characterization. To enrich the LAB promoter library, this study focused on the known strong constitutive promoter P₂₃ in LAB. Through error-prone PCR and dNTP analog-induced random mutagenesis, a library of 247 mutants of P₂₃ was generated by using the red fluorescent protein (RFP) fluorescence intensity as a high-throughput screening indicator in Streptococcus thermophilus. The activity of P₂₃ mutants varied from 0.01 to 3.63 times that of P₂₃. Similar trends of promoter strength were observed in Lactobacillus plantarum and Lactococcus lactis, but significant differences in Escherichia coli, indicating the library's specificity to LAB. To assess the application potential of this P₂₃ library, seven promoters with different strengths (0.28-2.58) were selected. The mutant promoters significantly enhanced the enzyme activities of superoxide dismutase (SOD), β-glucuronidase (GusA), and β-galactosidase (β-gal) in S. thermophilus. Notably, the mutant P_23-203 expressing SOD exhibited an enzyme activity of 382.9 U/mg, which was 1.65 times higher than the control (P₂₃). Similarly, the expression of GusA and β-gal were 1.82 and 1.28 times higher than those of P₂₃, respectively. This study provided a set of significantly different P₂₃ constitutive promoter mutant elements for the first time, laying the foundation for metabolic engineering and synthetic biology applications in LAB.