Meadows Corey W, Mingardon Florence, Garabedian Brett M, Baidoo Edward E K, Benites Veronica T, Rodrigues Andria V, Abourjeily Raya, Chanal Angelique, Lee Taek Soon
1Joint BioEnergy Institute, 5885 Hollis Street, 4th floor, Emeryville, CA 94608 USA.
2Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA.
Biotechnol Biofuels. 2018 Dec 28;11:340. doi: 10.1186/s13068-018-1342-2. eCollection 2018.
Geranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo.
In this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenable conditions for microbial mesophilic growth in conventional hosts such as or 31 putative GGRs were selected to test for potential reductase activity in vitro on farnesyl pyrophosphate, geranylgeranyl pyrophosphate, farnesol (FOH), and geranylgeraniol (GGOH). We report the discovery of several novel GGRs exhibiting significant activity toward various polyprenyl substrates under mild conditions (i.e., pH 7.4, = 37 °C), including the discovery of a novel bacterial GGR isolated from . In addition, we uncover new mechanistic insights within several GGR variants, including GGR-mediated phosphatase activity toward polyprenyl pyrophosphates and the first demonstration of completely hydrogenated GGOH and FOH substrates.
These collective results enhance the potential for metabolic engineers to manufacture a variety of isoprenoid-based biofuels, polymers, and chemical feedstocks in common microbial hosts such as or
香叶基香叶基还原酶（GGR）是一种含黄素的氧化还原酶，可氢化多种未活化的聚异戊二烯底物，这些底物大多在古菌中进一步用于脂质生物合成或在植物中用于叶绿素生物合成。迄今为止，仅有少数GGR基因在体外或体内被证实可还原聚异戊二烯底物。
在本研究中，我们旨在通过寻找在诸如大肠杆菌或枯草芽孢杆菌等传统宿主中适合微生物嗜温生长的条件下发挥作用的新基因，来扩展已确认的GGR活性范围。选择了31个假定的GGR来测试其在体外对法呢基焦磷酸、香叶基香叶基焦磷酸、法呢醇（FOH）和香叶基香叶醇（GGOH）的潜在还原酶活性。我们报告了发现几种新型GGR，它们在温和条件下（即pH 7.4，温度=37°C）对各种聚异戊二烯底物表现出显著活性，包括从嗜热栖热菌中分离出的一种新型细菌GGR。此外，我们还揭示了几种GGR变体的新机制见解，包括GGR对聚异戊二烯焦磷酸的磷酸酶活性以及首次证明完全氢化的GGOH和FOH底物。
这些综合结果增强了代谢工程师在诸如大肠杆菌或枯草芽孢杆菌等常见微生物宿主中生产各种基于异戊二烯类的生物燃料、聚合物和化学原料的潜力。
