Department of Life Science, Research Institute for Natural Sciences, Hanyang University, 222, Wangsimni-ro, Seongdong-gu, Seoul, 04763, Republic of Korea.
Arca Eir, C-323, Daedeok Biz Center, 17 Techno4-ro, Yuseong-gu, Daejeon, 34013, Republic of Korea.
Microb Cell Fact. 2020 Nov 30;19(1):220. doi: 10.1186/s12934-020-01480-4.
Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin.
In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of α-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L) and content (7.28 mg g) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L.
A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.
玉米黄质是一种主要的叶黄素类色素，作为视网膜色素和抗氧化剂具有重要作用。由于玉米黄质有助于预防年龄相关性黄斑变性，因此其在个性化营养和药物应用中的商业用途不断扩大。为了满足个性化治疗和药物应用的定量要求，有必要生产高纯度的玉米黄质。
在这项研究中，为了满足工业应用的定量要求，我们通过 CRISPR-Cas9 核糖核蛋白介导的基因敲入系统生成了一个双敲除突变体。番茄红素 ε 环化酶 (LCYE) 的编辑导致叶黄素生物合成的 α 支链在玉米黄质环氧化酶基因 (ZEP) 的敲除突变体中被消除。双敲除突变体 (dzl) 在培养 3 天后，玉米黄质的产量 (5.24 mg/L) 和含量 (7.28 mg/g) 比亲本株系高出 60%。此外，通过培养基优化，将 dzl 突变体的 3 天玉米黄质产量提高到 6.84 mg/L。
使用 CRISPR-Cas9 通过基因编辑成功开发出一种消除叶黄素生产的衣藻株系。本研究为克服高纯度玉米黄质生产的下游工艺难题提供了一种解决方案。
