用于筛选各种单萜合酶文库的自动化管道。,Scientific Reports

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用于筛选各种单萜合酶文库的自动化管道。
Scientific Reports ( IF 3.8 ) Pub Date : 2019-08-15 , DOI: 10.1038/s41598-019-48452-2
Nicole G H Leferink ₁ , Mark S Dunstan ₁ , Katherine A Hollywood ₁ , Neil Swainston ₁ , Andrew Currin ₁ , Adrian J Jervis ₁ , Eriko Takano ₁ , Nigel S Scrutton ₁

Affiliation

单萜类化合物是结构上不同的一组天然产物，其用途包括药物，调味剂，香料，农药和生物燃料。通过将异源类异戊二烯生产途径引入工程微生物，合成生物学的最新进展为实现这种化学多样性提供了新途径。由于支化反应机理的性质，单萜合酶在单萜类化合物生产平台中表达时通常会产生多种产物。由于蛋白序列与催化的环化反应之间缺乏相关性，因此萜烯合酶的合理工程设计具有挑战性。由于需要有限的先验序列功能知识，定向进化提供了一种有吸引力的蛋白质工程替代策略。然而，缺乏用于检测多种挥发性萜烯产物的便捷高通量筛选分析方法，阻碍了萜烯合酶的直接进化。在这里，我们应用了自动流水线来筛选各种单萜合酶文库，采用了结合了GC-MS的机器人液体处理平台和自动数据提取功能。我们使用管道来筛选to烯合酶变体文库，该文库具有在三个可塑性区域中发生突变的能力，能够生产多种单萜产物。我们成功地确定了具有变化的产品概况的变体，并证明了自动筛选结果与传统摇瓶培养物之间的良好一致性。此外，获得了有关pin烯合酶催化的环化反应的有用见解，包括确定具有最高可塑性的位置，以及区域2在碳正离子环化中的意义。获得的结果将有助于预测和设计新型萜烯合酶对清洁的单萜类产品的活性。

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An automated pipeline for the screening of diverse monoterpene synthase libraries.

Monoterpenoids are a structurally diverse group of natural products with applications as pharmaceuticals, flavourings, fragrances, pesticides, and biofuels. Recent advances in synthetic biology offer new routes to this chemical diversity through the introduction of heterologous isoprenoid production pathways into engineered microorganisms. Due to the nature of the branched reaction mechanism, monoterpene synthases often produce multiple products when expressed in monoterpenoid production platforms. Rational engineering of terpene synthases is challenging due to a lack of correlation between protein sequence and cyclisation reaction catalysed. Directed evolution offers an attractive alternative protein engineering strategy as limited prior sequence-function knowledge is required. However, directed evolution of terpene synthases is hampered by the lack of a convenient high-throughput screening assay for the detection of multiple volatile terpene products. Here we applied an automated pipeline for the screening of diverse monoterpene synthase libraries, employing robotic liquid handling platforms coupled to GC-MS, and automated data extraction. We used the pipeline to screen pinene synthase variant libraries, with mutations in three areas of plasticity, capable of producing multiple monoterpene products. We successfully identified variants with altered product profiles and demonstrated good agreement between the results of the automated screen and traditional shake-flask cultures. In addition, useful insights into the cyclisation reaction catalysed by pinene synthase were obtained, including the identification of positions with the highest level of plasticity, and the significance of region 2 in carbocation cyclisation. The results obtained will aid the prediction and design of novel terpene synthase activities towards clean monoterpenoid products.

更新日期：2019-08-15

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