Wenyi Sun1,2,3, Xiaobing Yang2, Xueying Wang2, Xinping Lin4, Yanan Wang2, Sufang Zhang2, Yushi Luan1, Zongbao K Zhao5. 1. School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, China. 2. Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China. 3. School of Life Sciences, Jilin Normal University, 1301 Haifeng Street, Siping, 136000, China. 4. School of Food Science and Technology, Dalian Polytechnic University, 1 Qinggongyuan, Dalian, 116034, China. 5. Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, China. zhaozb@dicp.ac.cn.
Abstract
OBJECTIVES: 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.
OBJECTIVES: To target a carotenoid biosynthetic gene in the oleaginous yeastRhodosporidium 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.
Authors: Joonhoon Kim; Samuel T Coradetti; Young-Mo Kim; Yuqian Gao; Junko Yaegashi; Jeremy D Zucker; Nathalie Munoz; Erika M Zink; Kristin E Burnum-Johnson; Scott E Baker; Blake A Simmons; Jeffrey M Skerker; John M Gladden; Jon K Magnuson Journal: Front Bioeng Biotechnol Date: 2021-01-08
Authors: Han Ming Gan; Bolaji N Thomas; Nicole T Cavanaugh; Grace H Morales; Ashley N Mayers; Michael A Savka; André O Hudson Journal: PeerJ Date: 2017-11-14 Impact factor: 2.984