A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase
In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116...
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Frontiers Media S.A.
2022-01-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2021.770302/full |
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| author | Feng Zhou Yan Xu Xiaoqing Mu Xiaoqing Mu Yao Nie |
| author_facet | Feng Zhou Yan Xu Xiaoqing Mu Xiaoqing Mu Yao Nie |
| author_sort | Feng Zhou |
| collection | DOAJ |
| description | In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the kcat/Km of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product. |
| first_indexed | 2024-04-11T20:42:42Z |
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| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-11T20:42:42Z |
| publishDate | 2022-01-01 |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-9692bcae8f584b7493ca233dc683a62e2022-12-22T04:04:10ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-01-01910.3389/fbioe.2021.770302770302A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate DehydrogenaseFeng Zhou0Yan Xu1Xiaoqing Mu2Xiaoqing Mu3Yao Nie4Lab of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, ChinaLab of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, ChinaLab of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, ChinaInstitute of Industrial Technology, Suqian Jiangnan University, Suqian, ChinaLab of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, ChinaIn this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the kcat/Km of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.https://www.frontiersin.org/articles/10.3389/fbioe.2021.770302/fulllevulinic acidreductive aminationglutamate dehydrogenasestructureguided protein engineering(R)-4-aminopentanoic acid |
| spellingShingle | Feng Zhou Yan Xu Xiaoqing Mu Xiaoqing Mu Yao Nie A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase Frontiers in Bioengineering and Biotechnology levulinic acid reductive amination glutamate dehydrogenase structureguided protein engineering (R)-4-aminopentanoic acid |
| title | A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase |
| title_full | A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase |
| title_fullStr | A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase |
| title_full_unstemmed | A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase |
| title_short | A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase |
| title_sort | sustainable approach for synthesizing r 4 aminopentanoic acid from levulinic acid catalyzed by structure guided tailored glutamate dehydrogenase |
| topic | levulinic acid reductive amination glutamate dehydrogenase structureguided protein engineering (R)-4-aminopentanoic acid |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2021.770302/full |
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