Sun Wenyi, Yang Xiaobing, Wang Xueying, Lin Xinping, Wang Yanan, Zhang Sufang, Luan Yushi, Zhao Zongbao K
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.
Biotechnol Lett. 2017 Jul;39(7):1001-1007. doi: 10.1007/s10529-017-2324-3. Epub 2017 Mar 23.
To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.
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.
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.
通过农杆菌介导的转化（AMT）方法靶向产油酵母红冬孢酵母中的类胡萝卜素生物合成基因。
从红冬孢酵母NP11的基因组DNA中扩增出先前被指定编码类胡萝卜素生物合成基因CRTI的RHTO_04602位点，并将其克隆到二元质粒pZPK-mcs中，得到pZPK-CRT。利用无限制克隆策略将HYG表达盒插入pZPK-CRT的CRTI序列中。将所得质粒按照AMT方法用于转化红冬孢酵母细胞，得到了一些白色转化子。对这些转化子的测序分析证实了CRTI的同源重组和插入失活。当用CRTI表达盒转化白色变体时，细胞变红并像野生型菌株NP11一样产生类胡萝卜素。
成功对CrtI位点进行同源靶向证实了RHTO_04602在红冬孢酵母类胡萝卜素生物合成中的功能。它为这种非模式酵母物种的代谢工程提供了有价值的信息。
