The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450_cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450_cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.

使用细胞色素 P450 酶对萜烯进行功能化是一种生产有用衍生物的通用途径，这些衍生物可以进一步转化为增值产品。许多萜烯具有疏水性和挥发性，使得它们作为 P450 酶底物的可用性在微生物生产过程中受到显着限制。在这项研究中，我们开发了一种策略，通过将萜烯合酶和 P450 连接在一起来提高萜烯分子对 P450 反应的可及性。作为模型系统，研究了 1,8-桉树脑合成酶 (CS) 和 P450 _cin的融合蛋白，结果表明单萜类 1,8-桉树脑的羟基化提高了 5.4 倍。_{CS-P450 cin}的结构分析SEC-SAXS 的融合蛋白表明二聚体形成具有两个结构域的活性位点的优选方向。我们还将酶融合策略应用于氧化倍半萜烯表异辛烯，并且融合酶显着改善了工程大肠杆菌中的白黄酮醇生产。通过对融合策略正反例的分析，我们提出了基于结构的融合酶预测和评价的关键因素。开发用于萜烯合酶和 P450 的融合酶为疏水性萜烯化合物的氧化提供了一种有效的策略。该策略可广泛应用于提高疏水萜烯中间体功能化产物的生物合成效价。