Pylb-based overexpression of cytochrome P450 in Bacillus subtilis 168

Inducer-free expression systems are promising tools for biorefinery because they can reduce the reliance on inducers, reducing production costs and simplifying processes. Owing to their broad range of substrate structures and catalytic reactions, cytochrome P450s are promising biocatalysts to produce value-added compounds. However, unsuitable levels of cytochrome P450 expression could result in cell stress, affecting the efficiency of the biocatalyst. Here, we assessed the potential of Pylb, a reported growth-phase-dependent promoter derived from Bacillus subtilis 168, to develop an inducer-free expression system, especially cytochrome P450 expression, in B. subtilis, a key workhorse strain. Utilizing a green fluorescent protein (GFP) reporter, we observed differential expression patterns under the control of Pylb and the constitutive promoter P43 in recombinant Escherichia coli and B. subtilis. Recombinant B. subtilis cultivated at 37 °C showed 2.8-fold higher bacterial fluorescence compared to cultivation at 30 °C. Codon-optimized engineered P450-BM3, which can convert octane to octanols, was selected as a model cytochrome P450 in this study. In the Pylb-based system, the expression of cytochrome P450 in recombinant B. subtilis can be detected at 24 h and increases over time as shown by the purpald assay. The activity of the overexpressed P450 was confirmed by the conversion of octane to octanols. Within one hour, the resting cells of recombinant B. subtilis produced 0.15 ± 0.04 mM of 1-octanol and 0.31 ± 0.08 mM of 2-octanol. Overall, the inducer-free Pylb-based system developed here is a potential biocatalyst for biorefinery applications.