Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering
The demand for L-tryptophan (L-Trp) has been rapidly increasing across various industries, including pharmaceuticals, food, and animal feed. However, traditional production methods have been unable to efficiently meet this growing demand. Hence, this study aimed to develop strategies for enhancing L...
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2025-05-01
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| Series: | Fermentation |
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| Online Access: | https://www.mdpi.com/2311-5637/11/5/267 |
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| author | Zhenghao Gao Fengli Wu Zhidan Zhang Xu Zhang Yuansen Hu Qinhong Wang Shuaibing Zhang |
| author_facet | Zhenghao Gao Fengli Wu Zhidan Zhang Xu Zhang Yuansen Hu Qinhong Wang Shuaibing Zhang |
| author_sort | Zhenghao Gao |
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| description | The demand for L-tryptophan (L-Trp) has been rapidly increasing across various industries, including pharmaceuticals, food, and animal feed. However, traditional production methods have been unable to efficiently meet this growing demand. Hence, this study aimed to develop strategies for enhancing L-Trp production in <i>Escherichia coli</i>. Firstly, an L-Trp-producing strain was selected and subjected to atmospheric and room temperature plasma (ARTP) mutagenesis to generate a mutant library. This was followed by high-throughput screening using an L-Trp-specific riboswitch and a yellow fluorescent protein (YFP)-based biosensor in a flow cytometric cell sorting (FACS) system. Among the screened mutants, GT3938 exhibited a 1.94-fold increase in L-Trp production. Subsequently, rational metabolic engineering was applied to GT3938 by knocking out the L-Trp intracellular transporter gene (<i>tnaB</i>), enhancing the expression of the aromatic amino acid exporter (YddG) and optimizing precursor supply pathways. The resulting strain, zh08, achieved an L-Trp titer of 3.05 g/L in shake-flask fermentation, representing a 7.71-fold improvement over the original strain. This study demonstrated an effective strategy for industrial strain development by integrating biosensor-assisted, high-throughput screening with rational metabolic engineering. |
| format | Article |
| id | doaj-art-c14f6c5014ca416290d9a9f21d29e7bf |
| institution | Kabale University |
| issn | 2311-5637 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
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| series | Fermentation |
| spelling | doaj-art-c14f6c5014ca416290d9a9f21d29e7bf2025-08-20T03:47:48ZengMDPI AGFermentation2311-56372025-05-0111526710.3390/fermentation11050267Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic EngineeringZhenghao Gao0Fengli Wu1Zhidan Zhang2Xu Zhang3Yuansen Hu4Qinhong Wang5Shuaibing Zhang6School of Biological Engineering, Henan University of Technology, Zhengzhou 450001, ChinaState Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaState Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaState Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaSchool of Biological Engineering, Henan University of Technology, Zhengzhou 450001, ChinaState Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaSchool of Biological Engineering, Henan University of Technology, Zhengzhou 450001, ChinaThe demand for L-tryptophan (L-Trp) has been rapidly increasing across various industries, including pharmaceuticals, food, and animal feed. However, traditional production methods have been unable to efficiently meet this growing demand. Hence, this study aimed to develop strategies for enhancing L-Trp production in <i>Escherichia coli</i>. Firstly, an L-Trp-producing strain was selected and subjected to atmospheric and room temperature plasma (ARTP) mutagenesis to generate a mutant library. This was followed by high-throughput screening using an L-Trp-specific riboswitch and a yellow fluorescent protein (YFP)-based biosensor in a flow cytometric cell sorting (FACS) system. Among the screened mutants, GT3938 exhibited a 1.94-fold increase in L-Trp production. Subsequently, rational metabolic engineering was applied to GT3938 by knocking out the L-Trp intracellular transporter gene (<i>tnaB</i>), enhancing the expression of the aromatic amino acid exporter (YddG) and optimizing precursor supply pathways. The resulting strain, zh08, achieved an L-Trp titer of 3.05 g/L in shake-flask fermentation, representing a 7.71-fold improvement over the original strain. This study demonstrated an effective strategy for industrial strain development by integrating biosensor-assisted, high-throughput screening with rational metabolic engineering.https://www.mdpi.com/2311-5637/11/5/267<i>Escherichia coli</i>L-Tryptophanbiosensorhigh-throughput screeningmetabolic engineeringARTP mutagenesis |
| spellingShingle | Zhenghao Gao Fengli Wu Zhidan Zhang Xu Zhang Yuansen Hu Qinhong Wang Shuaibing Zhang Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering Fermentation <i>Escherichia coli</i> L-Tryptophan biosensor high-throughput screening metabolic engineering ARTP mutagenesis |
| title | Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering |
| title_full | Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering |
| title_fullStr | Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering |
| title_full_unstemmed | Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering |
| title_short | Improvement of L-Tryptophan Production in <i>Escherichia coli</i> Using Biosensor-Based, High-Throughput Screening and Metabolic Engineering |
| title_sort | improvement of l tryptophan production in i escherichia coli i using biosensor based high throughput screening and metabolic engineering |
| topic | <i>Escherichia coli</i> L-Tryptophan biosensor high-throughput screening metabolic engineering ARTP mutagenesis |
| url | https://www.mdpi.com/2311-5637/11/5/267 |
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