Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation
Uridine diphosphate glycosyltransferases (UGTs) as fine catalysts of glycosylation are increasingly used in the synthesis of natural products. Sucrose synthase (SuSy) is recognized as a powerful tool for in situ regenerating sugar donors for the UGT-catalyzed reaction. It is crucial to select the ap...
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MDPI AG
2022-04-01
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| Series: | Applied Sciences |
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| author | Yehui Tao Ping Sun Ruxin Cai Yan Li Honghua Jia |
| author_facet | Yehui Tao Ping Sun Ruxin Cai Yan Li Honghua Jia |
| author_sort | Yehui Tao |
| collection | DOAJ |
| description | Uridine diphosphate glycosyltransferases (UGTs) as fine catalysts of glycosylation are increasingly used in the synthesis of natural products. Sucrose synthase (SuSy) is recognized as a powerful tool for in situ regenerating sugar donors for the UGT-catalyzed reaction. It is crucial to select the appropriate SuSy for cooperation with UGT in a suitable way. In the present study, eukaryotic SuSy from <i>Arabidopsis</i><i>thaliana</i> (<i>At</i>SUS1) helped <i>stevia</i> glycosyltransferase UGT76G1 achieve the complete conversion of stevioside (30 g/L) into rebaudioside A (RebA). Position of the individual transcription units containing the genes encoding <i>At</i>SUS1 and UGT76G1 in the expression plasmid has an effect, but less than that of the fusion order of these genes on RebA yield. Fusion of the <i>C</i>-terminal of <i>At</i>SUS1 and the <i>N</i>-terminal of UGT76G1 with rigid linkers are conducive to maintaining enzyme activities. When the same fusion strategy was applied to a L637M-T640V double mutant of prokaryotic SuSy from <i>Acidithiobacillus caldus</i> (<i>Ac</i>SuSym), 18.8 ± 0.6 g/L RebA (a yield of 78.2%) was accumulated in the reaction mixture catalyzed by the fusion protein Acm-R3-76G1 (the <i>C</i>-terminal of <i>Ac</i>SuSym and the <i>N</i>-terminal of UGT76G1 were linked with (EAAAK)3). This work would hopefully reveal the potential of UGT-SuSy fusion in improving the cascade enzymatic glycosylation. |
| first_indexed | 2024-03-09T11:11:47Z |
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| language | English |
| last_indexed | 2024-03-09T11:11:47Z |
| publishDate | 2022-04-01 |
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| series | Applied Sciences |
| spelling | doaj.art-2ea1c5df08524c0593bdf607873768362023-12-01T00:41:25ZengMDPI AGApplied Sciences2076-34172022-04-01128391110.3390/app12083911Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic BiotransformationYehui Tao0Ping Sun1Ruxin Cai2Yan Li3Honghua Jia4College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, ChinaCollege of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, ChinaCollege of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, ChinaCollege of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, ChinaCollege of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, ChinaUridine diphosphate glycosyltransferases (UGTs) as fine catalysts of glycosylation are increasingly used in the synthesis of natural products. Sucrose synthase (SuSy) is recognized as a powerful tool for in situ regenerating sugar donors for the UGT-catalyzed reaction. It is crucial to select the appropriate SuSy for cooperation with UGT in a suitable way. In the present study, eukaryotic SuSy from <i>Arabidopsis</i><i>thaliana</i> (<i>At</i>SUS1) helped <i>stevia</i> glycosyltransferase UGT76G1 achieve the complete conversion of stevioside (30 g/L) into rebaudioside A (RebA). Position of the individual transcription units containing the genes encoding <i>At</i>SUS1 and UGT76G1 in the expression plasmid has an effect, but less than that of the fusion order of these genes on RebA yield. Fusion of the <i>C</i>-terminal of <i>At</i>SUS1 and the <i>N</i>-terminal of UGT76G1 with rigid linkers are conducive to maintaining enzyme activities. When the same fusion strategy was applied to a L637M-T640V double mutant of prokaryotic SuSy from <i>Acidithiobacillus caldus</i> (<i>Ac</i>SuSym), 18.8 ± 0.6 g/L RebA (a yield of 78.2%) was accumulated in the reaction mixture catalyzed by the fusion protein Acm-R3-76G1 (the <i>C</i>-terminal of <i>Ac</i>SuSym and the <i>N</i>-terminal of UGT76G1 were linked with (EAAAK)3). This work would hopefully reveal the potential of UGT-SuSy fusion in improving the cascade enzymatic glycosylation.https://www.mdpi.com/2076-3417/12/8/3911glycosyltransferase UGT76G1rebaudioside Astevioside<i>Stevia rebaudiana</i>sucrose synthase |
| spellingShingle | Yehui Tao Ping Sun Ruxin Cai Yan Li Honghua Jia Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation Applied Sciences glycosyltransferase UGT76G1 rebaudioside A stevioside <i>Stevia rebaudiana</i> sucrose synthase |
| title | Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation |
| title_full | Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation |
| title_fullStr | Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation |
| title_full_unstemmed | Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation |
| title_short | Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation |
| title_sort | exploring the strategy of fusing sucrose synthase to glycosyltransferase ugt76g1 in enzymatic biotransformation |
| topic | glycosyltransferase UGT76G1 rebaudioside A stevioside <i>Stevia rebaudiana</i> sucrose synthase |
| url | https://www.mdpi.com/2076-3417/12/8/3911 |
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