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...
Guardado en:
| Autores principales: | , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/6d85d240568f47b98f39c64a954417c9 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| Sumario: | 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. |
|---|