转基因
表情盒
生物
清脆的
Cas9
突变体
绿色荧光蛋白
基因
遗传筛选
八氢番茄红素脱氢酶
基因沉默
分子生物学
细胞生物学
遗传学
重组DNA
载体(分子生物学)
作者
Ae Jin Ryu,Byeong‐ryool Jeong,Nak Heon Kang,Sang Goo Jeon,Min Gi Sohn,Hyo Jin Yun,Jong Min Lim,Sun Young Jeong,Youn‐Il Park,Won Joong Jeong,Sunghoon Park,Yong Keun Chang,Ki Jun Jeong
标识
DOI:10.1016/j.biortech.2021.125676
摘要
Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.
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