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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Autores principales:	Márcia R. Couto, Joana L. Rodrigues, Lígia R. Rodrigues
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	UDP-glucose dehydrogenase UDP-glucuronic acid glycosaminoglycans biosynthesis heterologous production <i>Escherichia coli</i> <i>Saccharomyces cerevisiae</i> Science Q
Acceso en línea:	https://doaj.org/article/6d85d240568f47b98f39c64a954417c9
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spelling	oai:doaj.org-article:6d85d240568f47b98f39c64a954417c92021-11-25T18:11:07ZCloning, Expression and Characterization of UDP-Glucose Dehydrogenases10.3390/life111112012075-1729https://doaj.org/article/6d85d240568f47b98f39c64a954417c92021-11-01T00:00:00Zhttps://www.mdpi.com/2075-1729/11/11/1201https://doaj.org/toc/2075-1729Uridine 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.Márcia R. CoutoJoana L. RodriguesLígia R. RodriguesMDPI AGarticleUDP-glucose dehydrogenaseUDP-glucuronic acidglycosaminoglycans biosynthesisheterologous production<i>Escherichia coli</i><i>Saccharomyces cerevisiae</i>ScienceQENLife, Vol 11, Iss 1201, p 1201 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	UDP-glucose dehydrogenase UDP-glucuronic acid glycosaminoglycans biosynthesis heterologous production <i>Escherichia coli</i> <i>Saccharomyces cerevisiae</i> Science Q
spellingShingle	UDP-glucose dehydrogenase UDP-glucuronic acid glycosaminoglycans biosynthesis heterologous production <i>Escherichia coli</i> <i>Saccharomyces cerevisiae</i> Science Q Márcia R. Couto Joana L. Rodrigues Lígia R. Rodrigues Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases
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.
format	article
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
title	Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases
title_short	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_sort	cloning, expression and characterization of udp-glucose dehydrogenases
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/6d85d240568f47b98f39c64a954417c9
work_keys_str_mv	AT marciarcouto cloningexpressionandcharacterizationofudpglucosedehydrogenases AT joanalrodrigues cloningexpressionandcharacterizationofudpglucosedehydrogenases AT ligiarrodrigues cloningexpressionandcharacterizationofudpglucosedehydrogenases
_version_	1718411501860028416

Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

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