Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides

被引:13
|
作者
Sun, Wenyi [1 ,2 ]
Yang, Xiaobing [1 ]
Wang, Xueying [1 ]
Jiao, Xiang [1 ]
Zhang, Sufang [1 ]
Luan, Yushi [2 ]
Zhao, Zongbao K. [1 ]
机构
[1] Chinese Acad Sci, Dalian Inst Chem Phys, Div Biotechnol, 457 Zhongshan Rd, Dalian 116023, Peoples R China
[2] Dalian Univ Technol, Sch Life Sci & Biotechnol, Dalian, Peoples R China
基金
中国国家自然科学基金;
关键词
FLP/FRT system; Homologous recombination; Inducible expression; Nuclear localization signal sequence; Rhodosporidium toruloides; EXCISION; SYSTEM; GENOME; GENES; FLP;
D O I
10.1007/s10529-018-2542-3
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Objectives To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides. Results Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium. Conclusions We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.
引用
收藏
页码:933 / 940
页数:8
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