Qi Shen, Fang Yan, Ya-Wen Li, Jian Wang, Jia Ji, Wen-Xin Yan, Dan-Chen He, Ping Song, Tian-Qiong Shi
{"title":"结合 \"金门 \"和 CRISPR 技术,扩展用于脂溶性亚罗威氏菌的 YALIcloneHR 工具包","authors":"Qi Shen, Fang Yan, Ya-Wen Li, Jian Wang, Jia Ji, Wen-Xin Yan, Dan-Chen He, Ping Song, Tian-Qiong Shi","doi":"10.1007/s10529-023-03444-1","DOIUrl":null,"url":null,"abstract":"<p>Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of <i>Yarrowia lipolytica</i>. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene <i>PEX10</i> was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in <i>Y. lipolytica</i>, which would help produce other chemicals derived from acetyl-CoA in the future.</p>","PeriodicalId":2,"journal":{"name":"ACS Applied Bio Materials","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2023-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology\",\"authors\":\"Qi Shen, Fang Yan, Ya-Wen Li, Jian Wang, Jia Ji, Wen-Xin Yan, Dan-Chen He, Ping Song, Tian-Qiong Shi\",\"doi\":\"10.1007/s10529-023-03444-1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of <i>Yarrowia lipolytica</i>. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene <i>PEX10</i> was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in <i>Y. lipolytica</i>, which would help produce other chemicals derived from acetyl-CoA in the future.</p>\",\"PeriodicalId\":2,\"journal\":{\"name\":\"ACS Applied Bio Materials\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2023-12-08\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Applied Bio Materials\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1007/s10529-023-03444-1\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MATERIALS SCIENCE, BIOMATERIALS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Applied Bio Materials","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s10529-023-03444-1","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MATERIALS SCIENCE, BIOMATERIALS","Score":null,"Total":0}
引用次数: 0
摘要
酵母代谢工程是提高细胞工厂生产能力的关键方法。要获得基因稳定的重组菌株,外源 DNA 最好能整合到基因组中。此前,我们开发了一个金门工具包 YALIcloneNHEJ,它可以作为一个高效的模块化克隆工具包,通过脂肪分解亚罗酵母的先天非同源末端连接修复机制随机整合多基因通路。我们利用 CRISPR 技术设计了更多的同源臂构件,从而扩展了该工具包。因此,重建的工具包被命名为 YALIcloneHR,设计用于基因特异性敲除和整合。为了验证该系统的有效性,我们选择了 PEX10 基因作为基因敲除的靶标。该系统随后被应用于花生四烯酸的生产,重建后的菌株可积累 4.8%的花生四烯酸。该工具包将扩展脂溶性酵母菌的基因编辑技术,这将有助于将来生产其他由乙酰-CoA衍生的化学物质。
Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology
Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.