Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.
OBJECTIVE
To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.


RESULTS
The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.


CONCLUSIONS
Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.
School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, China.|Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China.|School of Life Sciences, Jilin Normal University, 1301 Haifeng Street, Siping, 136000, China.
Wenyi
W
Sun
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China.
Xiaobing
X
Yang
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China.
Xueying
X
Wang
School of Food Science and Technology, Dalian Polytechnic University, 1 Qinggongyuan, Dalian, 116034, China.
Xinping
X
Lin
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China.
Yanan
Y
Wang
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China.
Sufang
S
Zhang
School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, China.
Yushi
Y
Luan
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China. zhaozb@dicp.ac.cn.
Zongbao K
ZK
Zhao
D060054:Agrobacterium / Q000235:genetics*; D053898:Biosynthetic Pathways / Q000235:genetics*; D002338:Carotenoids / Q000096:biosynthesis*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D059765:Homologous Recombination; D012248:Rhodotorula / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D002338:Carotenoids
Agrobacterium-mediated transformation; Carotenoids; Homologous gene targeting; Rhodosporidium toruloides