Gonzalez-Esquer C Raul, Ferlez Bryan, Weraduwage Sarathi M, Kirst Henning, Lantz Alexandra T, Turmo Aiko, Sharkey Thomas D, Kerfeld Cheryl A
MSU-DOE Plant Research Laboratory, Michigan State University East Lansing MI 48824 USA
Department of Biochemistry & Molecular Biology, Michigan State University East Lansing MI 48824 USA.
RSC Adv. 2021 Sep 8;11(48):29997-30005. doi: 10.1039/d1ra05710c. eCollection 2021 Sep 6.
Terpene synthases are biotechnologically-relevant enzymes with a variety of applications. However, they are typically poor catalysts and have been difficult to engineer. Structurally, most terpene synthases share two conserved domains (α- and β-domains). Some also contain a third domain containing a second active site (γ-domain). Based on the three-domain architecture, we hypothesized that αβ terpene synthases could be engineered by insertion of a heterologous domain at the site of the γ-domain (an approach we term "Insertion-engineering terpene synthase"; Ie-TS). We demonstrate that by mimicking the domain architecture of αβγ terpene synthases, we can redesign isoprene synthase (ISPS), an αβ terpene synthase, while preserving enzymatic activity. Insertion of GFP or a SpyCatcher domain within ISPS introduced new functionality while maintaining or increasing catalytic turnover. This insertion-engineering approach establishes that the γ-domain position is accessible for incorporation of additional sequence features and enables the rational engineering of terpene synthases for biotechnology.
萜烯合酶是具有多种应用的与生物技术相关的酶。然而，它们通常是较差的催化剂，并且难以进行工程改造。在结构上，大多数萜烯合酶共享两个保守结构域（α-和β-结构域）。有些还包含第三个结构域，其中含有第二个活性位点（γ-结构域）。基于这种三结构域架构，我们推测αβ萜烯合酶可以通过在γ-结构域的位置插入异源结构域来进行工程改造（我们将这种方法称为“插入工程萜烯合酶”；Ie-TS）。我们证明，通过模仿αβγ萜烯合酶的结构域架构，我们可以重新设计异戊二烯合酶（ISPS），一种αβ萜烯合酶，同时保留酶活性。在ISPS中插入绿色荧光蛋白（GFP）或一个SpyCatcher结构域引入了新功能，同时保持或提高了催化周转率。这种插入工程方法表明，γ-结构域位置可用于并入额外的序列特征，并能够对用于生物技术的萜烯合酶进行合理工程改造。
