Biodiversity Research Center, Academia Sinica, No. 128 Academia Road, Sec. 2, Nankang, Taipei 115, Taiwan; Department of Life Sciences, National Chung Hsing University, No. 250, Kuo Kuang Rd, Taichung 402, Taiwan.
Department of Medical Research, China Medical University Hospital, China Medical University, No. 91 Hsueh-Shih Road, Taichung 402, Taiwan.
Bioresour Technol. 2017 Dec;245(Pt A):899-905. doi: 10.1016/j.biortech.2017.07.116. Epub 2017 Jul 23.
In this study, an astaxanthin-biosynthesis Kluyveromyces marxianus strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes Hpchyb and bkt was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of Hpchyb and bkt. To improve the yield further, the gene Hpchyb from Haematococcus pluvialis was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination. Using repeated-integration approach of bkt and the mutated Hpchyb into Sm23, the S3-2 strain was obtained and shown to produce the 3S, 3'S-astaxanthin at 9972µg/g DCW in a 5L fermentor.
本研究首次构建了一株能生产 31µg/g DCW 虾青素的毕赤酵母 Sm23 工程菌，随后通过重复基因组整合虾青素生物合成关键基因 Hpchyb 和 bkt 来提高基因拷贝数和虾青素产量。得到了四个改良菌株，虾青素产量和菌株中类胡萝卜素总产量随 Hpchyb 和 bkt 的拷贝数增加而增加。为了进一步提高产量，对来自雨生红球藻的基因 Hpchyb 进行了定点突变修饰，以提高酶效率或/和防止异源蛋白通过泛素化降解。通过 bkt 和突变的 Hpchyb 的重复整合方法，将其整合到 Sm23 中，得到了 S3-2 菌株，在 5L 发酵罐中以 9972µg/g DCW 的产量生产 3S,3'S-虾青素。
