Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides.
The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation (ATMT) protocol for effective gene integration into the R. toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R. toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals.
Dalian Institute of Chemical Physics, CAS, Dalian, China; University of Chinese Academy of Sciences, Beijing, China.
Xinping
X
Lin
Yanan
Y
Wang
Sufang
S
Zhang
Zhiwei
Z
Zhu
Yongjin J
YJ
Zhou
Fan
F
Yang
Wenyi
W
Sun
Xueying
X
Wang
Zongbao K
ZK
Zhao
D060054:Agrobacterium / Q000235:genetics; D001487:Basidiomycota / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D016681:Genome, Fungal; D060847:Metabolic Engineering / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D011995:Recombination, Genetic
Agrobacterium-mediated transformation; Rhodosporidium toruloides; antibiotic resistance; gene integration