DECHEMA Research Institute, Industrial Biotechnology, Frankfurt am Main, 60486, Germany.
Johann Wolfgang Goethe-University, Faculty of Biological Sciences, Frankfurt am Main, 60323, Germany.
ACS Synth Biol. 2020 May 15;9(5):981-986. doi: 10.1021/acssynbio.9b00432. Epub 2020 May 4.
Terpenes constitute the largest class of natural products with more than 70 000 compounds. Many different terpenes find applications in the flavor and fragrance industry or can be used as fine chemicals or drugs. In some bacteria, noncanonical terpenes with 11 carbon atoms are synthesized via a GPP-C2-methyltransferase and the subsequent conversion of 2-methyl-GPP by certain terpene synthases into mainly 2-methylisoborneol and 2-methylenebornane. Many other C-terpenes were reported as side products, but they are synthesized only in minor amounts by the bacterial C-terpene biosynthesis pathway. To enable biotechnological synthesis of these largely unexplored natural products, we changed the product selectivity of the 2-methylenebornane synthase from by a semirational protein engineering approach. Active site amino acids with impact on the product selectivity were identified and variants with completely altered product spectra could be identified and characterized. The gathered data provide new insights into the structure-function relationship for C-terpene synthases and demonstrate the production of formerly inaccessible noncanonical terpenes.
萜类化合物构成了最大的天然产物类别，拥有超过 70000 种化合物。许多不同的萜类化合物在香料和香精行业有应用，或者可以用作精细化学品或药物。在某些细菌中，具有 11 个碳原子的非典型萜类化合物通过 GPP-C2-甲基转移酶合成，随后由某些萜烯合酶将 2-甲基-GPP 转化为主要的 2-甲基异莰醇和 2-亚甲基降冰片烷。许多其他 C-萜烯被报道为副产物，但它们仅由细菌 C-萜烯生物合成途径以少量合成。为了能够对这些在很大程度上尚未开发的天然产物进行生物技术合成，我们通过半理性的蛋白质工程方法改变了 2-亚甲基降冰片烷合酶的产物选择性。鉴定出对产物选择性有影响的活性位点氨基酸，并可以鉴定和表征具有完全改变的产物谱的变体。收集的数据提供了萜烯合酶结构-功能关系的新见解，并证明了以前无法获得的非典型萜类化合物的生产。
