CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae
CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae
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DOI：
10.1007/s10295-016-1831-x
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发表时间：
2016-11-01
影响因子：
3.4
通讯作者：
Kim, Hyo Jin
中科院分区：
文献类型：
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作者：
Chin, Young-Wook;Kang, Woo-Kyung;Kim, Hyo Jin
近几十年来，基因组编辑技术取得了巨大进步。在新兴的基因组编辑技术中，CRISPR/Cas9被认为是革命性的，因为它易于使用，并且在目标生物中编辑基因的精度很高。CRISPR/Cas9技术也被用于去除不利的靶基因。在本研究中，我们利用CRISPR/Cas9技术对酵母在乙醇发酵过程中形成的潜在致癌物氨基甲酸乙酯（EC）进行了还原。由于酵母编码精氨酸酶的CAR1基因是形成氨基甲酸酯的关键基因，我们利用CRISPR/Cas9技术通过完全缺失该基因或在CAR1位点引入无义突变来灭活酵母CAR1基因。工程酵母菌的精氨酸酶比活性降低了98%，而乙醇发酵性能与之相当。此外，与亲本酵母菌株相比，car1失活突变体显示EC和尿素的形成减少。重要的是，CRISPR/Cas9技术能够在不从载体上留下外源基因残余的情况下产生car1失活的酵母菌株，这表明通过CRISPR/Cas9技术改造的酵母菌可能会避开转基因生物的调控。
Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.