Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc. 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki, 210-8681, Japan.
Ajinomoto-Genetika Research Institute, 1st Dorozhny pr. 1-1, Moscow, 117545, Russian Federation.
J Biotechnol. 2020 Dec 20;324:21-27. doi: 10.1016/j.jbiotec.2020.09.021. Epub 2020 Sep 24.
Linalool is a monoterpene alcohol, which imparts floral scents to a variety of plants and is extensively used in various kinds of products, such as processed foods and beverages for fragrances and flavors. However, linalool from natural resources is racemate, and production of linalool enantiomers is difficult. To produce stereospecific linalool, we evaluated linalool synthase genes (LINS) from plants, such as Actinidia arguta (AaLINS) and Coriandrum sativum (CsLINS) for (S)-specific LINS and a gram-positive bacterium Streptomyces clavuligerus (ScLINS) for (R)-specific LINS, with Pantoea ananatis strain as the host. Among the 16 LINS examined, AaLINS and ScLINS showed the best (S)-linalool production and (R)-linalool production, respectively, with 100 % enantio excess. Co-expression of the mutated farnesyl diphosphate synthase gene, ispA* (S80 F), from Escherichia coli along with the LINS genes also improved linalool production. In order to prevent volatilization and cell toxicity of linalool, two-phase cultivation with isopropyl myristate was done, which had positive effects on linalool production. The carbon flux to the MVA pathway from glucose was increased by inactivating a membrane-bound glucose dehydrogenase. Overall, 5.60 g/L (S)-linalool and 3.71 g/L (R)-linalool were produced from 60.0 g/L glucose by introduction of AaLINS-ispA* and ScLINS-ispA* in P. ananatis, respectively.
芳樟醇是一种单萜醇，它为多种植物赋予花香，并广泛用于各种产品，如加工食品和饮料中的香料和风味。然而，天然资源中的芳樟醇是外消旋体，且其对映异构体的生产较为困难。为了生产立体专一的芳樟醇，我们评估了植物中的芳樟醇合酶基因（LINS），如软枣猕猴桃（AaLINS）和芫荽（CsLINS）用于（S）-特异性 LINS 和革兰氏阳性细菌棒状链霉菌（ScLINS）用于（R）-特异性 LINS，以 Pantoea ananatis 菌株作为宿主。在所研究的 16 个 LINS 中，AaLINS 和 ScLINS 分别表现出最佳的（S）-芳樟醇生产和（R）-芳樟醇生产，具有 100%的对映体过量。与 LINS 基因共表达的大肠杆菌中的突变法呢基二磷酸合酶基因，ispA*（S80F），也提高了芳樟醇的产量。为了防止芳樟醇的挥发和细胞毒性，进行了两相培养，使用了肉豆蔻酸异丙酯，这对芳樟醇的生产有积极影响。通过使膜结合葡萄糖脱氢酶失活，从葡萄糖到 MVA 途径的碳通量增加。总的来说，通过在 P. ananatis 中引入 AaLINS-ispA* 和 ScLINS-ispA*，从 60.0g/L 葡萄糖中分别生产了 5.60g/L（S）-芳樟醇和 3.71g/L（R）-芳樟醇。
