T Rehka, Lin Fu, Chen Xixian, Zhang Congqiang
Singapore Institute of Food and Biotechnology Innovation (SIFBI), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore.
Adv Biotechnol (Singap). 2025 Jan 13;3(1):3. doi: 10.1007/s44307-024-00056-2.
Enzymes are the cornerstone of biocatalysis, biosynthesis and synthetic biology. However, their applicability is often limited by low substrate selectivity. A prime example is the bifunctional linalool/nerolidol synthase (LNS) that can use both geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) to produce linalool and nerolidol, respectively. This bifunctionality can lead to undesired byproducts in synthetic biology applications. To enhance enzyme specificity and create monofunctional linalool synthases, we modified amino acids in the loop between helices C and D of four bifunctional mushroom LNSs. Through these modifications, we successfully shifted the substrate preference of two LNSs (ApLNS from Agrocybe pediades and HsLNS from Hypholoma sublateritium) from FPP towards GPP. Although complete monofunctionality was not achieved, we significantly increased linalool yield by 13 times while minimizing nerolidol production to 1% of the wildtype HsLNS. Docking simulations revealed a substantial reduction in the FPP binding score compared to that of the wildtype. Molecular dynamics simulations suggested that Tyr300 in the apo HsLNS mutant has a greater tendency to adopt an inward orientation. Together with Met77, the inward-facing Tyr300 creates a steric barrier that prevents the longer FPP molecule from entering the substrate binding pocket, thereby greatly reducing its activity towards FPP. This study demonstrates the potential of enzyme engineering to design substrate-specific terpene synthases, which is a challenging task and few successful examples are available. The insights gained can inform future enzyme design efforts, including the development of artificial intelligence models.
酶是生物催化、生物合成和合成生物学的基石。然而，它们的适用性常常受到低底物选择性的限制。一个典型的例子是双功能芳樟醇/橙花叔醇合酶（LNS），它可以分别利用香叶基二磷酸（GPP）和法尼基二磷酸（FPP）生成芳樟醇和橙花叔醇。这种双功能性在合成生物学应用中可能会导致不需要的副产物。为了提高酶的特异性并创建单功能芳樟醇合酶，我们对四种双功能蘑菇LNSs的C螺旋和D螺旋之间的环中的氨基酸进行了修饰。通过这些修饰，我们成功地将两种LNSs（来自嗜热栖热放线菌的ApLNS和来自亚侧耳的HsLNS）的底物偏好从FPP转向了GPP。尽管没有实现完全的单功能性，但我们将芳樟醇产量显著提高了13倍，同时将橙花叔醇的产量降至野生型HsLNS的1%。对接模拟显示，与野生型相比，FPP结合分数大幅降低。分子动力学模拟表明，apo HsLNS突变体中的Tyr300更倾向于向内取向。与Met77一起，向内的Tyr300形成了一个空间位垒，阻止较长的FPP分子进入底物结合口袋，从而大大降低了其对FPP的活性。这项研究证明了酶工程在设计底物特异性萜类合酶方面的潜力，这是一项具有挑战性的任务，成功的例子很少。所获得的见解可以为未来的酶设计工作提供参考，包括人工智能模型的开发。
