High-level soluble production of firefly luciferase in E. coli via promoter engineering

Firefly luciferase (FLuc) is a key enzyme catalyzing bioluminescent reactions. Owing to its strong penetrability of bioluminescence, low background signal, high catalytic efficiency, high sensitivity and specificity for substrates, FLuc is widely used in food safety, cosmetics, environmental monitoring and biomedicine However, the poor soluble expression and low productivity of FLuc limit its practical applications. Although we have achieved heterologous expression of FLuc using T7 expression system in E. coli, the proportion of inclusion bodies remained high. Therefore, we investigated promoter engineering on FLuc expression levels. First, we screened the 21 endogenous promoters in E. coli. Among which the pgi promoter showed superior potential for regulating protein expression. Subsequently, through random mutation to the -35 box of the pgi promoter, identification of functional mutation sites and optimization of fermentation conditions, the yield of FLuc increased to 0.3 mg/mL. This represented a 354% increase over the initial yield and a 52.06% increase in soluble proportion, marking the highest expression level reported to date. Our optimization strategy not only enhances the soluble expression of recombinant FLuc in E. coli, but also avoids the use of IPTG, thereby laying a foundation for the industrial application of FLuc.