Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes
Abstract Background Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5′-diph...
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| Format: | Article |
| Language: | English |
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BMC
2019-07-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-019-1168-z |
| _version_ | 1819026782899142656 |
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| author | Dan-Hua Meng Ran-Ran Du Lu-Zhou Chen Meng-Ting Li Fei Liu Jin Hou Yi-Kang Shi Feng-Shan Wang Ju-Zheng Sheng |
| author_facet | Dan-Hua Meng Ran-Ran Du Lu-Zhou Chen Meng-Ting Li Fei Liu Jin Hou Yi-Kang Shi Feng-Shan Wang Ju-Zheng Sheng |
| author_sort | Dan-Hua Meng |
| collection | DOAJ |
| description | Abstract Background Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5′-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. Results To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L−1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. Conclusions This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA. |
| first_indexed | 2024-12-21T05:32:03Z |
| format | Article |
| id | doaj.art-ce20b51ec4a646778b14350724328e20 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-21T05:32:03Z |
| publishDate | 2019-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-ce20b51ec4a646778b14350724328e202022-12-21T19:14:31ZengBMCMicrobial Cell Factories1475-28592019-07-0118111110.1186/s12934-019-1168-zCascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymesDan-Hua Meng0Ran-Ran Du1Lu-Zhou Chen2Meng-Ting Li3Fei Liu4Jin Hou5Yi-Kang Shi6Feng-Shan Wang7Ju-Zheng Sheng8Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong UniversityKey Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong UniversityKey Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong UniversityKey Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong UniversityKey Laboratory of Biopharmaceuticals, Shandong Academy of Pharmaceutical SciencesState Key Laboratory of Microbiology, Shandong UniversityNational Glycoengineering Research Center, Shandong UniversityKey Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong UniversityKey Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong UniversityAbstract Background Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5′-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. Results To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L−1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. Conclusions This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.http://link.springer.com/article/10.1186/s12934-019-1168-zBiocatalysisWhole cell synthesisNucleotide sugarUDP-GlcAHyperthermophilic enzymeNAD+ regeneration |
| spellingShingle | Dan-Hua Meng Ran-Ran Du Lu-Zhou Chen Meng-Ting Li Fei Liu Jin Hou Yi-Kang Shi Feng-Shan Wang Ju-Zheng Sheng Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes Microbial Cell Factories Biocatalysis Whole cell synthesis Nucleotide sugar UDP-GlcA Hyperthermophilic enzyme NAD+ regeneration |
| title | Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes |
| title_full | Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes |
| title_fullStr | Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes |
| title_full_unstemmed | Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes |
| title_short | Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes |
| title_sort | cascade synthesis of uridine 5 diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes |
| topic | Biocatalysis Whole cell synthesis Nucleotide sugar UDP-GlcA Hyperthermophilic enzyme NAD+ regeneration |
| url | http://link.springer.com/article/10.1186/s12934-019-1168-z |
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