An active site normally locates inside of enzymes, substrates should go through the tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450BsβHI (Q85H/ V170I) derived from hydroxylase P450Bsβ from Bacillus subtilis was chosen as study model, which is reported as a potential decarbonylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was

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... rformed for modulating the substrate preference and improving the decarbonylase activity of P450BsβHI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.