Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli
Abstract Background With the development of metabolic engineering and synthetic biology, the biosynthesis of aromatic compounds has attracted much attention. Cinnamylamine is an aromatic compound derived from l-phenylalanine, which is used in the synthesis of biologically active molecules, including...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-09-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
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| Online Access: | https://doi.org/10.1186/s13068-022-02199-7 |
| _version_ | 1811200873819275264 |
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| author | Qi Wang Linlin Ma Zhiguo Wang Quan Chen Qian Wang Qingsheng Qi |
| author_facet | Qi Wang Linlin Ma Zhiguo Wang Quan Chen Qian Wang Qingsheng Qi |
| author_sort | Qi Wang |
| collection | DOAJ |
| description | Abstract Background With the development of metabolic engineering and synthetic biology, the biosynthesis of aromatic compounds has attracted much attention. Cinnamylamine is an aromatic compound derived from l-phenylalanine, which is used in the synthesis of biologically active molecules, including drugs, and energetic materials. Cinnamylamine has been mainly synthesized by chemical methods to date, and few reports have focused on the biosynthesis of cinnamylamine. Therefore, it is desirable to establish an efficient biosynthesis method for cinnamylamine. Results The ω-aminotransferase Cv-ωTA from Chromobacterium violaceum has been demonstrated to have high enzyme activity in the conversion of cinnamaldehyde to cinnamylamine. To prevent the preferable conversion of cinnamaldehyde to cinnamyl alcohol in wild-type Escherichia coli, the E. coli MG1655 strain with reduced aromatic aldehyde reduction (RARE) in which six aldehyde ketone reductase and alcohol dehydrogenase genes have been knocked out was employed. Then, the carboxylic acid reductase from Neurospora crassa (NcCAR) and phosphopantetheinyl transferase (PPTase) from E. coli were screened for a high conversion rate of cinnamic acid to cinnamaldehyde. To shift the equilibrium of the reaction toward cinnamylamine, saturation mutagenesis of Cv-ωTA at key amino acid residues was performed, and Cv-ωTA Y168G had the highest conversion rate with 88.56 mg/L cinnamylamine obtained after 4 h of fermentation. Finally, by optimizing the substrates and the supply of the cofactors, PLP and NADPH, in the fermentation, the yield of cinnamylamine in engineered E. coli reached 523.15 mg/L. Conclusion We achieved the first biosynthesis of cinnamylamine using cinnamic acid as the precursor in E. coli using a combinatorial metabolic engineering strategy. This study provides a reference for the biosynthesis of other amine compounds and lays a foundation for the de novo synthesis of cinnamylamine. |
| first_indexed | 2024-04-12T02:11:02Z |
| format | Article |
| id | doaj.art-3d5a1370d93d4825a8c1f7cadb737893 |
| institution | Directory Open Access Journal |
| issn | 2731-3654 |
| language | English |
| last_indexed | 2024-04-12T02:11:02Z |
| publishDate | 2022-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj.art-3d5a1370d93d4825a8c1f7cadb7378932022-12-22T03:52:23ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542022-09-0115111210.1186/s13068-022-02199-7Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coliQi Wang0Linlin Ma1Zhiguo Wang2Quan Chen3Qian Wang4Qingsheng Qi5State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong UniversityState Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong UniversityInstitute of Ageing Research, School of Medicine, Hangzhou Normal UniversityCAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of SciencesState Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong UniversityState Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong UniversityAbstract Background With the development of metabolic engineering and synthetic biology, the biosynthesis of aromatic compounds has attracted much attention. Cinnamylamine is an aromatic compound derived from l-phenylalanine, which is used in the synthesis of biologically active molecules, including drugs, and energetic materials. Cinnamylamine has been mainly synthesized by chemical methods to date, and few reports have focused on the biosynthesis of cinnamylamine. Therefore, it is desirable to establish an efficient biosynthesis method for cinnamylamine. Results The ω-aminotransferase Cv-ωTA from Chromobacterium violaceum has been demonstrated to have high enzyme activity in the conversion of cinnamaldehyde to cinnamylamine. To prevent the preferable conversion of cinnamaldehyde to cinnamyl alcohol in wild-type Escherichia coli, the E. coli MG1655 strain with reduced aromatic aldehyde reduction (RARE) in which six aldehyde ketone reductase and alcohol dehydrogenase genes have been knocked out was employed. Then, the carboxylic acid reductase from Neurospora crassa (NcCAR) and phosphopantetheinyl transferase (PPTase) from E. coli were screened for a high conversion rate of cinnamic acid to cinnamaldehyde. To shift the equilibrium of the reaction toward cinnamylamine, saturation mutagenesis of Cv-ωTA at key amino acid residues was performed, and Cv-ωTA Y168G had the highest conversion rate with 88.56 mg/L cinnamylamine obtained after 4 h of fermentation. Finally, by optimizing the substrates and the supply of the cofactors, PLP and NADPH, in the fermentation, the yield of cinnamylamine in engineered E. coli reached 523.15 mg/L. Conclusion We achieved the first biosynthesis of cinnamylamine using cinnamic acid as the precursor in E. coli using a combinatorial metabolic engineering strategy. This study provides a reference for the biosynthesis of other amine compounds and lays a foundation for the de novo synthesis of cinnamylamine.https://doi.org/10.1186/s13068-022-02199-7Cinnamylamine biosynthesisω-AminotransferaseMetabolic engineeringCinnamic acidEscherichia coli |
| spellingShingle | Qi Wang Linlin Ma Zhiguo Wang Quan Chen Qian Wang Qingsheng Qi Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli Biotechnology for Biofuels and Bioproducts Cinnamylamine biosynthesis ω-Aminotransferase Metabolic engineering Cinnamic acid Escherichia coli |
| title | Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli |
| title_full | Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli |
| title_fullStr | Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli |
| title_full_unstemmed | Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli |
| title_short | Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli |
| title_sort | construction and yield optimization of a cinnamylamine biosynthesis route in escherichia coli |
| topic | Cinnamylamine biosynthesis ω-Aminotransferase Metabolic engineering Cinnamic acid Escherichia coli |
| url | https://doi.org/10.1186/s13068-022-02199-7 |
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