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

• The T1.2RQ lipase gene was successfully expressed in the photosynthetic bacterium Cereibacter sphaeroides , establishing this organism as a viable and potentially energy-efficient platform for recombinant enzyme production. • The puf promoter (oxygen-regulated) demonstrated superior performance compared to the lac promoter under anaerobic photoheterotrophic conditions, confirming its effectiveness for driving high-level expression when oxygen tension is low. • The expressed T1.2RQ lipase displayed high thermostability (Optimal 65°C) and activity on long-chain substrates, suitable for industrial applications. 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.