Institutes of Physical Science and Information Technology, Anhui University, Hefei 230000, China.
School of Life Science and Technology, Dalian University, Dalian 116000, China.
Biomed Res Int. 2021 Mar 17;2021:6653500. doi: 10.1155/2021/6653500. eCollection 2021.
Pinene, a natural active monoterpene, is widely used as a flavoring agent, perfume, medicine, and biofuel. Although genetically engineered microorganisms have successfully produced pinene, to date, the biological yield of pinene is much lower than that of semiterpenes (isoprene) and sesquiterpenes (farnesene). In addition to the low heterologous expression of geranyl pyrophosphate synthase (GPPS) and pinene synthase (PS), cytotoxicity due to accumulation of the monoterpene also limits the production of pinene in microorganisms. In this study, we attempted to use two strategies to increase the biological yield of pinene. By deleting the random coils of GPPS and PS alone or in combination, a strain with a 335% yield increase was obtained. Additionally, upon computer-guided molecular modeling and docking of GPPS with isopentenyl pyrophosphate (IPP), its substrate, the key sites located within the catalytic pocket for substrate binding, was predicted. After screening, a strain harboring the T273R mutation of GPPS was selected among a batch of mutations of the key sites with a 154% increase in pinene yield.
蒎烯是一种天然的活性单萜，被广泛用作香料、香水、药物和生物燃料。尽管基因工程微生物已成功生产出蒎烯，但迄今为止，蒎烯的生物产量远低于半萜（异戊二烯）和倍半萜（法呢烯）。除了香叶基焦磷酸合酶（GPPS）和蒎烯合酶（PS）的异源表达水平较低外，由于单萜的积累而产生的细胞毒性也限制了微生物中蒎烯的生产。在这项研究中，我们试图使用两种策略来提高蒎烯的生物产量。通过单独或组合删除 GPPS 和 PS 的无规卷曲，获得了产量提高 335%的菌株。此外，通过对 GPPS 与异戊烯焦磷酸（IPP，其底物）进行计算机引导的分子建模和对接，预测了位于催化口袋中用于底物结合的关键位点。经过筛选，从关键位点的一批突变体中选择了一株携带 GPPS 的 T273R 突变的菌株，蒎烯产量提高了 154%。
