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Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer
BACKGROUND: Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric aci...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935971/ https://www.ncbi.nlm.nih.gov/pubmed/29729665 http://dx.doi.org/10.1186/s12934-018-0914-y |
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author | Chen, Na Wang, Jingya Zhao, Yunying Deng, Yu |
author_facet | Chen, Na Wang, Jingya Zhao, Yunying Deng, Yu |
author_sort | Chen, Na |
collection | PubMed |
description | BACKGROUND: Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. RESULTS: A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae. CONCLUSIONS: In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0914-y) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5935971 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-59359712018-05-11 Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer Chen, Na Wang, Jingya Zhao, Yunying Deng, Yu Microb Cell Fact Research BACKGROUND: Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. RESULTS: A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae. CONCLUSIONS: In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0914-y) contains supplementary material, which is available to authorized users. BioMed Central 2018-05-05 /pmc/articles/PMC5935971/ /pubmed/29729665 http://dx.doi.org/10.1186/s12934-018-0914-y Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Chen, Na Wang, Jingya Zhao, Yunying Deng, Yu Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
title | Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
title_full | Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
title_fullStr | Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
title_full_unstemmed | Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
title_short | Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
title_sort | metabolic engineering of saccharomyces cerevisiae for efficient production of glucaric acid at high titer |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935971/ https://www.ncbi.nlm.nih.gov/pubmed/29729665 http://dx.doi.org/10.1186/s12934-018-0914-y |
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