Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine
Abstract Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has sho...
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| Format: | Article |
| Language: | English |
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BMC
2021-01-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-020-01501-2 |
| _version_ | 1818562711536009216 |
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| author | Langxing Liao Yonghui Zhang Yali Wang Yousi Fu Aihui Zhang Ruodian Qiu Shuhao Yang Baishan Fang |
| author_facet | Langxing Liao Yonghui Zhang Yali Wang Yousi Fu Aihui Zhang Ruodian Qiu Shuhao Yang Baishan Fang |
| author_sort | Langxing Liao |
| collection | DOAJ |
| description | Abstract Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle. |
| first_indexed | 2024-12-14T01:07:21Z |
| format | Article |
| id | doaj.art-5b8c0d466c6c4dd2949f8bf29cf96bf0 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-14T01:07:21Z |
| publishDate | 2021-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-5b8c0d466c6c4dd2949f8bf29cf96bf02022-12-21T23:22:55ZengBMCMicrobial Cell Factories1475-28592021-01-0120111210.1186/s12934-020-01501-2Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucineLangxing Liao0Yonghui Zhang1Yali Wang2Yousi Fu3Aihui Zhang4Ruodian Qiu5Shuhao Yang6Baishan Fang7Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityDepartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniversityAbstract Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle.https://doi.org/10.1186/s12934-020-01501-2l-tert-leucineGlucose dehydrogenaseLeucine dehydrogenaseFusion enzymeWhole cells |
| spellingShingle | Langxing Liao Yonghui Zhang Yali Wang Yousi Fu Aihui Zhang Ruodian Qiu Shuhao Yang Baishan Fang Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine Microbial Cell Factories l-tert-leucine Glucose dehydrogenase Leucine dehydrogenase Fusion enzyme Whole cells |
| title | Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine |
| title_full | Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine |
| title_fullStr | Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine |
| title_full_unstemmed | Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine |
| title_short | Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine |
| title_sort | construction and characterization of a novel glucose dehydrogenase leucine dehydrogenase fusion enzyme for the biosynthesis of l tert leucine |
| topic | l-tert-leucine Glucose dehydrogenase Leucine dehydrogenase Fusion enzyme Whole cells |
| url | https://doi.org/10.1186/s12934-020-01501-2 |
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