Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System
Theanine is a non-protein amino acid that is highly represented in tea plants and is one of the delicious ingredients in tea. In recent years, the global market demand for theanine has continued to rise, and the industry has developed rapidly. Here, we designed and constructed a promising pathway in...
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| Format: | Article |
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MDPI AG
2023-09-01
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| Series: | Fermentation |
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| Online Access: | https://www.mdpi.com/2311-5637/9/10/875 |
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| author | Ruiqi Cao Shunyang Hu Yao Lu Wei Wang Zhongdan Fu Jie Cheng |
| author_facet | Ruiqi Cao Shunyang Hu Yao Lu Wei Wang Zhongdan Fu Jie Cheng |
| author_sort | Ruiqi Cao |
| collection | DOAJ |
| description | Theanine is a non-protein amino acid that is highly represented in tea plants and is one of the delicious ingredients in tea. In recent years, the global market demand for theanine has continued to rise, and the industry has developed rapidly. Here, we designed and constructed a promising pathway in <i>Escherichia coli</i> to produce L-theanine. This biosynthesis pathway employs four enzymes to achieve the production of L-theanine. This route involves the co-expression of four functional enzymes: γ-glutamylmethylamide synthetase (GMAS) from <i>Methyloversatilis</i> universalis, polyphosphate kinase (PPK) from <i>E. coli</i>, alanine transaminase from <i>Bacillus subtilis</i> (BsAld), and alanine decarboxylase from <i>Camellia sinensis</i> (CsAlaDC). Polyphosphate kinase from <i>Escherichia coli</i> was overexpressed in <i>E. coli</i> FD02, constructing an ATP regeneration system that increased the titer of L-theanine by 13.4% compared to <i>E. coli</i> FD01. A titer of 334 mg/L of L-theanine was produced via engineering strain FD03 in shake flasks. Moreover, glutamine permease from <i>Saccharomyces cereviside</i> (GNP1) was overexpressed in <i>E. coli</i> FD04, and the L-theanine titer increased by 14.7%. Finally, 2.9 g/L of L-theanine was obtained via FD04 in a 1 L bioreactor. In addition, the molecular docking results indicated that L-glutamate could bind to the hydrophobic cavity of GMAS due to the formation of hydrogen bonds and hydrophobic interactions with the surrounding amino acid residues. |
| first_indexed | 2024-03-10T21:15:41Z |
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| issn | 2311-5637 |
| language | English |
| last_indexed | 2024-03-10T21:15:41Z |
| publishDate | 2023-09-01 |
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| record_format | Article |
| series | Fermentation |
| spelling | doaj.art-63be143d7d3944db8a14f35c0d2e07a72023-11-19T16:26:00ZengMDPI AGFermentation2311-56372023-09-0191087510.3390/fermentation9100875Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration SystemRuiqi Cao0Shunyang Hu1Yao Lu2Wei Wang3Zhongdan Fu4Jie Cheng5Meat Processing Key Laboratory of Sichuan Province, College of Food and Biological Engineering, Chengdu University, Chengdu 610106, ChinaMeat Processing Key Laboratory of Sichuan Province, College of Food and Biological Engineering, Chengdu University, Chengdu 610106, ChinaCollege of Biological and Chemical Engineering, Guangxi University of Science and Technology, Liuzhou 545006, ChinaMeat Processing Key Laboratory of Sichuan Province, College of Food and Biological Engineering, Chengdu University, Chengdu 610106, ChinaMeat Processing Key Laboratory of Sichuan Province, College of Food and Biological Engineering, Chengdu University, Chengdu 610106, ChinaMeat Processing Key Laboratory of Sichuan Province, College of Food and Biological Engineering, Chengdu University, Chengdu 610106, ChinaTheanine is a non-protein amino acid that is highly represented in tea plants and is one of the delicious ingredients in tea. In recent years, the global market demand for theanine has continued to rise, and the industry has developed rapidly. Here, we designed and constructed a promising pathway in <i>Escherichia coli</i> to produce L-theanine. This biosynthesis pathway employs four enzymes to achieve the production of L-theanine. This route involves the co-expression of four functional enzymes: γ-glutamylmethylamide synthetase (GMAS) from <i>Methyloversatilis</i> universalis, polyphosphate kinase (PPK) from <i>E. coli</i>, alanine transaminase from <i>Bacillus subtilis</i> (BsAld), and alanine decarboxylase from <i>Camellia sinensis</i> (CsAlaDC). Polyphosphate kinase from <i>Escherichia coli</i> was overexpressed in <i>E. coli</i> FD02, constructing an ATP regeneration system that increased the titer of L-theanine by 13.4% compared to <i>E. coli</i> FD01. A titer of 334 mg/L of L-theanine was produced via engineering strain FD03 in shake flasks. Moreover, glutamine permease from <i>Saccharomyces cereviside</i> (GNP1) was overexpressed in <i>E. coli</i> FD04, and the L-theanine titer increased by 14.7%. Finally, 2.9 g/L of L-theanine was obtained via FD04 in a 1 L bioreactor. In addition, the molecular docking results indicated that L-glutamate could bind to the hydrophobic cavity of GMAS due to the formation of hydrogen bonds and hydrophobic interactions with the surrounding amino acid residues.https://www.mdpi.com/2311-5637/9/10/875L-theanineγ-glutamylmethylamine synthasealanine decarboxylaseATP regeneration systemtransporter |
| spellingShingle | Ruiqi Cao Shunyang Hu Yao Lu Wei Wang Zhongdan Fu Jie Cheng Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System Fermentation L-theanine γ-glutamylmethylamine synthase alanine decarboxylase ATP regeneration system transporter |
| title | Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System |
| title_full | Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System |
| title_fullStr | Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System |
| title_full_unstemmed | Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System |
| title_short | Fermentative Production of L-Theanine in <i>Escherichia coli</i> via the Construction of an Adenosine Triphosphate Regeneration System |
| title_sort | fermentative production of l theanine in i escherichia coli i via the construction of an adenosine triphosphate regeneration system |
| topic | L-theanine γ-glutamylmethylamine synthase alanine decarboxylase ATP regeneration system transporter |
| url | https://www.mdpi.com/2311-5637/9/10/875 |
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