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>

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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Main Authors: Zijing Zhu, Shuaihu Fang, Pingping Huang, Dianqiang Luo, Xiaobao Qi
Format: Article
Language:English
Published: MDPI AG 2025-06-01
Series:Fermentation
Subjects:
2-phenylethanol
<i>Saccharomyces cerevisiae</i>
CRISPRa
Ehrlich pathway
Online Access:https://www.mdpi.com/2311-5637/11/6/345
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Summary: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>.
ISSN:2311-5637

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