Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases
Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been con...
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MDPI AG
2021-11-01
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| Series: | Life |
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| author | Márcia R. Couto Joana L. Rodrigues Lígia R. Rodrigues |
| author_facet | Márcia R. Couto Joana L. Rodrigues Lígia R. Rodrigues |
| author_sort | Márcia R. Couto |
| collection | DOAJ |
| description | Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from <i>Zymomonas mobilis</i> (<i>Zm</i>UGD) and from <i>Lactobacillus johnsonii</i> (<i>Lbj</i>UGD) were expressed in <i>Escherichia coli</i>. These two enzymes and an additional eukaryotic one from <i>Capra hircus</i> (<i>Ch</i>UGD) were also expressed in <i>Saccharomyces cerevisiae</i> strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered <i>E. coli</i> strains expressing <i>Zm</i>UGD and <i>Lbj</i>UGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using <i>S. cerevisiae</i> as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing <i>Zm</i>UGD (17.9 µM) or <i>Ch</i>UGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, <i>E. coli</i> cell extract containing <i>Lbj</i>UGD was able to produce about 1800 µM, while <i>Zm</i>UGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using <i>S. cerevisiae</i> CEN.PK2-1C_pSP-GM_<i>Lbj</i>UGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications. |
| first_indexed | 2024-03-10T05:20:42Z |
| format | Article |
| id | doaj.art-6d85d240568f47b98f39c64a954417c9 |
| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-10T05:20:42Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
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| spelling | doaj.art-6d85d240568f47b98f39c64a954417c92023-11-23T00:04:21ZengMDPI AGLife2075-17292021-11-011111120110.3390/life11111201Cloning, Expression and Characterization of UDP-Glucose DehydrogenasesMárcia R. Couto0Joana L. Rodrigues1Lígia R. Rodrigues2Centre of Biological Engineering, University of Minho, 4710-057 Braga, PortugalCentre of Biological Engineering, University of Minho, 4710-057 Braga, PortugalCentre of Biological Engineering, University of Minho, 4710-057 Braga, PortugalUridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from <i>Zymomonas mobilis</i> (<i>Zm</i>UGD) and from <i>Lactobacillus johnsonii</i> (<i>Lbj</i>UGD) were expressed in <i>Escherichia coli</i>. These two enzymes and an additional eukaryotic one from <i>Capra hircus</i> (<i>Ch</i>UGD) were also expressed in <i>Saccharomyces cerevisiae</i> strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered <i>E. coli</i> strains expressing <i>Zm</i>UGD and <i>Lbj</i>UGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using <i>S. cerevisiae</i> as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing <i>Zm</i>UGD (17.9 µM) or <i>Ch</i>UGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, <i>E. coli</i> cell extract containing <i>Lbj</i>UGD was able to produce about 1800 µM, while <i>Zm</i>UGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using <i>S. cerevisiae</i> CEN.PK2-1C_pSP-GM_<i>Lbj</i>UGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.https://www.mdpi.com/2075-1729/11/11/1201UDP-glucose dehydrogenaseUDP-glucuronic acidglycosaminoglycans biosynthesisheterologous production<i>Escherichia coli</i><i>Saccharomyces cerevisiae</i> |
| spellingShingle | Márcia R. Couto Joana L. Rodrigues Lígia R. Rodrigues Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases Life UDP-glucose dehydrogenase UDP-glucuronic acid glycosaminoglycans biosynthesis heterologous production <i>Escherichia coli</i> <i>Saccharomyces cerevisiae</i> |
| title | Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases |
| title_full | Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases |
| title_fullStr | Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases |
| title_full_unstemmed | Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases |
| title_short | Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases |
| title_sort | cloning expression and characterization of udp glucose dehydrogenases |
| topic | UDP-glucose dehydrogenase UDP-glucuronic acid glycosaminoglycans biosynthesis heterologous production <i>Escherichia coli</i> <i>Saccharomyces cerevisiae</i> |
| url | https://www.mdpi.com/2075-1729/11/11/1201 |
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