CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i>
2-phenylethanol (2-PE), a rose-like fragrance compound, is widely used in the food industry. Conventional chemical synthesis of 2-PE faces significant challenges due to environmental concerns and consumer preferences; thus, using <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae<...
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2025-06-01
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| author | Zijing Zhu Shuaihu Fang Pingping Huang Dianqiang Luo Xiaobao Qi |
| author_facet | Zijing Zhu Shuaihu Fang Pingping Huang Dianqiang Luo Xiaobao Qi |
| author_sort | Zijing Zhu |
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| description | 2-phenylethanol (2-PE), a rose-like fragrance compound, is widely used in the food industry. Conventional chemical synthesis of 2-PE faces significant challenges due to environmental concerns and consumer preferences; thus, using <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) for 2-PE biosynthesis has become a preferable option. This study aimed to develop a CRISPR activation (CRISPRa)-mediated <i>S. cerevisiae</i> engineered strain for efficient 2-PE biosynthesis by activating Ehrlich pathway key genes <i>ARO10</i>, <i>ARO80</i>, and <i>ADH2.</i> Three guide sequences (GSs) were designed for each gene <i>ARO10</i>, <i>ARO80</i>, and <i>ADH2</i>, and nine single-gene CRISPRa strains were constructed. Gene expression levels, 2-PE concentrations, and cell density were quantified using quantitative real-time PCR (qPCR), high-performance liquid chromatography (HPLC), and OD<sub>600</sub> measurement, respectively. The optimal GSs of <i>ARO10</i>, <i>ARO80</i>, and <i>ADH2</i> were selected based on 2-PE concentrations of corresponding strains. The triple-gene CRISPRa strain INVScI-<i>ARO10</i>-<i>ARO80</i>-<i>ADH2</i> achieved a 214.04 mg/L 2-PE titer after 48 h, representing a 77.62% increase over the control with no significant effect on cell growth. These findings demonstrate that CRISPRa-mediated multi-gene activation constitutes a robust strategy for engineering high-performance 2-PE production systems in <i>S. cerevisiae</i>. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-06-01 |
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| series | Fermentation |
| spelling | doaj-art-ee1faa792072447ba2507d125feddcdf2025-08-20T03:24:37ZengMDPI AGFermentation2311-56372025-06-0111634510.3390/fermentation11060345CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i>Zijing Zhu0Shuaihu Fang1Pingping Huang2Dianqiang Luo3Xiaobao Qi4College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China2-phenylethanol (2-PE), a rose-like fragrance compound, is widely used in the food industry. Conventional chemical synthesis of 2-PE faces significant challenges due to environmental concerns and consumer preferences; thus, using <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) for 2-PE biosynthesis has become a preferable option. This study aimed to develop a CRISPR activation (CRISPRa)-mediated <i>S. cerevisiae</i> engineered strain for efficient 2-PE biosynthesis by activating Ehrlich pathway key genes <i>ARO10</i>, <i>ARO80</i>, and <i>ADH2.</i> Three guide sequences (GSs) were designed for each gene <i>ARO10</i>, <i>ARO80</i>, and <i>ADH2</i>, and nine single-gene CRISPRa strains were constructed. Gene expression levels, 2-PE concentrations, and cell density were quantified using quantitative real-time PCR (qPCR), high-performance liquid chromatography (HPLC), and OD<sub>600</sub> measurement, respectively. The optimal GSs of <i>ARO10</i>, <i>ARO80</i>, and <i>ADH2</i> were selected based on 2-PE concentrations of corresponding strains. The triple-gene CRISPRa strain INVScI-<i>ARO10</i>-<i>ARO80</i>-<i>ADH2</i> achieved a 214.04 mg/L 2-PE titer after 48 h, representing a 77.62% increase over the control with no significant effect on cell growth. These findings demonstrate that CRISPRa-mediated multi-gene activation constitutes a robust strategy for engineering high-performance 2-PE production systems in <i>S. cerevisiae</i>.https://www.mdpi.com/2311-5637/11/6/3452-phenylethanol<i>Saccharomyces cerevisiae</i>CRISPRaEhrlich pathway |
| spellingShingle | Zijing Zhu Shuaihu Fang Pingping Huang Dianqiang Luo Xiaobao Qi CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i> Fermentation 2-phenylethanol <i>Saccharomyces cerevisiae</i> CRISPRa Ehrlich pathway |
| title | CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i> |
| title_full | CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i> |
| title_fullStr | CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i> |
| title_full_unstemmed | CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i> |
| title_short | CRISPRa-Mediated Triple-Gene Activation of <i>ARO10</i>, <i>ARO80,</i> and <i>ADH2</i> for Enhancing 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in <i>Saccharomyces cerevisiae</i> |
| title_sort | crispra mediated triple gene activation of i aro10 i i aro80 i and i adh2 i for enhancing 2 phenylethanol biosynthesis via the ehrlich pathway in i saccharomyces cerevisiae i |
| topic | 2-phenylethanol <i>Saccharomyces cerevisiae</i> CRISPRa Ehrlich pathway |
| url | https://www.mdpi.com/2311-5637/11/6/345 |
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