Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway
| Autor: | Cláudio Remedios Frazão, Yoann Malbert, Christopher M. Topham, Jean Marie François, Thomas Walther |
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| Přispěvatelé: | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Molecular Forces Consulting, Toulouse White Biotechnology (TWB), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), valorization service of the 'Toulouse Transfer Technology', ANR-14-CE06-0024,SYNPATHIC,Ingénierie robuste et évolution dirigée de voies metaboliques synthétiques par intégration de la microfluidique et de la génomique(2014), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Models Molecular homosérine [SDV.BIO]Life Sciences [q-bio]/Biotechnology Protein Conformation Lactate dehydrogenase A Homoserine malate dehydrogenase Dehydrogenase Reductase Biochemistry Malate dehydrogenase Substrate Specificity 03 medical and health sciences chemistry.chemical_compound education Butylene Glycols Molecular Biology chemistry.chemical_classification education.field_of_study Binding Sites 030102 biochemistry & molecular biology biology Escherichia coli Proteins Lactococcus lactis Reproducibility of Results protein engineering Cell Biology biology.organism_classification ingénierie enzymatique Turnover number Biosynthetic Pathways Alcohol Oxidoreductases Butyrates Kinetics enzyme 030104 developmental biology Enzyme chemistry Metabolic Engineering protéine Mutagenesis Site-Directed synthetic biology site-directed mutagenesis escherichia coli protein |
| Zdroj: | Biochemical Journal Biochemical Journal, Portland Press, 2018, 475 (23), pp.3887-3901. ⟨10.1042/BCJ20180765⟩ Biochemical Journal, 2018, 475 (23), pp.3887-3901. ⟨10.1042/BCJ20180765⟩ |
| ISSN: | 0264-6021 1470-8728 |
| DOI: | 10.1042/BCJ20180765⟩ |
| Popis: | A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine, composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction of 2-keto-4-hydroxybutyrate (OHB) into (L)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In this study, we used a rational approach to engineer an OHB reductase by using the cytosolic (L)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (L)-malate dehydrogenase and (L)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a step-wise manner into these regions of the protein. This rational engineering led to the production of a Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (k cat ) on OHB that was increased by more than 2,000 fold (from 0.03 up to 65.0 s -1 ), which turned out to be 7 fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies (k cat /K m ) between natural and synthetic OHB substrates (84 and 31 s -1 mM -1 , respectively). Shake-flask cultivation of an homoserine-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert homoserine to OHB resulted in 89 % increased DHB production as compared to our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis |
| Databáze: | OpenAIRE |
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