Yang_2025_Biomolecules_15_842

Our previous study demonstrated that an Escherichia coli heterologous secretion expression system, mediated by superfolder green fluorescent protein (sfGFP) mutants, significantly enhances recombinant lipase yield and reduces large-scale production costs. In this study, we identified mScarlet3, a fast-folding fluorescent protein, as another effective mediator of secretion expression in E. coli. A novel lipolytic enzyme, named LipHu6, was identified through sequence alignment. Secretion expression of LipHu6 was achieved by fusing mScarlet3 to either its N- or C-terminus. The specific activity of mScarlet3-LipHu6 reached 669,151.75 U/mmol, slightly surpassing that of LipHu6 alone (646,682.69 U/mmol) and markedly exceeding that of sfGFP((-15))-LipHu6 (492,432.39 U/mmol). Notably, N-terminal mScarlet3 fusion had no impact on LipHu6 hydrolytic activity toward short-chain p-nitrophenyl fatty acyl esters (C2-C8). In contrast, mScarlet3-LipHu6 exhibited approximately 1.5- and 1.7-fold increases in hydrolytic activity toward p-nitrophenyl palmitate (p-NPP, C16) and p-nitrophenyl stearate (p-NPS, C18), respectively. In conclusion, this study establishes a novel E. coli heterologous secretion expression system mediated by mScarlet3, offering a highly efficient and cost-effective strategy for the large-scale production of lipolytic enzymes.