Heterologous expression of lipase gene in Cereibacter sphaeroides 2.4.1 driven by an anaerobic Photosynthesis-Inducible promoter.

Cereibacter sphaeroides 2.4.1, a purple non-sulfur bacterium, is a metabolically versatile organism capable of shifting between aerobic respiration and anaerobic photoheterotrophy. This adaptability makes it an attractive platform for sustainable biocatalysis that harnesses solar energy to drive high-energy biochemical reactions. In this study, we evaluated C. sphaeroides as a heterologous expression host for the thermostable T1.2RQ lipase from Geobacillus stearothermophilus T1.2 using the broad-host-range vector pRK415. To exploit light-responsive regulation, expression driven by the anaerobic/microaerobic-inducible puf promoter was compared with that driven by the constitutive lac promoter. Under anaerobic conditions, puf-driven expression (0.333 ± 0.044 U/mg) exceeded lac-driven expression (0.108 ± 0.01 U/mg), whereas under aerobic conditions the lac promoter yielded higher activity (0.542 ± 0.001 U/mg) than the puf promoter (0.247 ± 0.010 U/mg). These results demonstrate that the use of an indigenous promoter does not necessarily correlate with proportional increases in enzymatic output. SDS-PAGE and zymogram analyses confirmed expression of the recombinant enzyme, showing a consistent molecular mass of ∼ 43 kDa. The lipase exhibited optimal activity at 65°C and retained ∼ 70% activity after 3 h at 50°C. Maximum activity occurred at pH 10.0, and the enzyme remained stable between pH 7.0-10.0, maintaining > 75% residual activity after 180 min. Substrate specificity assays revealed activity toward multiple p-nitrophenyl esters, with a preference for short-chain substrates, particularly pNP butyrate. Collectively, these findings identify the puf promoter as a promising regulatory element for light-driven protein expression in C. sphaeroides. Coupling enzyme production to photosynthetic metabolism provides a low-energy strategy for industrial biocatalysis.