Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i>
The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon sour...
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MDPI AG
2021-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/5/2266 |
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| author | Zamira E. Soto-Varela Gema Cabrera Agustin Romero Domingo Cantero Antonio Valle Jorge Bolivar |
| author_facet | Zamira E. Soto-Varela Gema Cabrera Agustin Romero Domingo Cantero Antonio Valle Jorge Bolivar |
| author_sort | Zamira E. Soto-Varela |
| collection | DOAJ |
| description | The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. <i>Escherichia coli</i> is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the <i>ppc</i> gene highlights its central role in growth and C4 production. |
| first_indexed | 2024-03-09T00:34:07Z |
| format | Article |
| id | doaj.art-f93aae7f53704017adfe06d392d0a010 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T00:34:07Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f93aae7f53704017adfe06d392d0a0102023-12-11T18:19:27ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01225226610.3390/ijms22052266Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i>Zamira E. Soto-Varela0Gema Cabrera1Agustin Romero2Domingo Cantero3Antonio Valle4Jorge Bolivar5Department of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Chemical Engineering and Food Technology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Chemical Engineering and Food Technology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainThe biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. <i>Escherichia coli</i> is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the <i>ppc</i> gene highlights its central role in growth and C4 production.https://www.mdpi.com/1422-0067/22/5/2266C4 compoundsmalatesuccinateglycerolTCA cycleanaplerotic enzymes |
| spellingShingle | Zamira E. Soto-Varela Gema Cabrera Agustin Romero Domingo Cantero Antonio Valle Jorge Bolivar Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i> International Journal of Molecular Sciences C4 compounds malate succinate glycerol TCA cycle anaplerotic enzymes |
| title | Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i> |
| title_full | Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i> |
| title_fullStr | Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i> |
| title_full_unstemmed | Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i> |
| title_short | Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in <i>Escherichia coli</i> |
| title_sort | identification of enzymatic bottlenecks for the aerobic production of malate from glycerol by the systematic gene overexpression of anaplerotic enzymes in i escherichia coli i |
| topic | C4 compounds malate succinate glycerol TCA cycle anaplerotic enzymes |
| url | https://www.mdpi.com/1422-0067/22/5/2266 |
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