Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides
Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides
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多个基因功能性整合到产油酵母红冬孢酵母基因组中
DOI：
10.1111/1567-1364.12140
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发表时间：
2014-06-01
影响因子：
3.2
通讯作者：
Zhao, Zongbao K.
中科院分区：
文献类型：
--
作者：
Lin, Xinping;Wang, Yanan;Zhao, Zongbao K.
球红冬孢酵母（Rhodosporidiumtoruloides）是一种产微生物脂类和类胡萝卜素的优良酵母。然而，这种非常规酵母的进一步合理工程化仍然具有挑战性，部分原因是缺乏有效和可靠的转化方法。在这项研究中，我们开发了一种农杆菌介导的转化（ATMT）协议，有效的基因整合到圆红曲霉基因组。通过菌落PCR、Western blot分析和基因组步移等方法，证实了单倍体和二倍体菌株的整合。我们进一步证明了多个基因可以通过连续的ATMT整合，导致工程菌株同时对多种抗生素具有抗性。本研究为圆红酵母外源基因的功能整合和表达提供了一种实用的方法，为开发遗传工具和构建生产生物燃料分子和生物化学品的上级菌株奠定了基础。
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.