Print Email Facebook Twitter Functional expression of a bacterial α-ketoglutarate dehydrogenase in the cytosol of Saccharomyces cerevisiae Title Functional expression of a bacterial α-ketoglutarate dehydrogenase in the cytosol of Saccharomyces cerevisiae Author Baldi, N. (TU Delft BT/Industriele Microbiologie) Dykstra, James C. (Student TU Delft) Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie) Pabst, Martin (TU Delft OLD BT/Cell Systems Engineering) Wu, Liang (DSM) Benjamin, Kirsten R. (Amyris Inc) Vente, André (DSM) Pronk, J.T. (TU Delft BT/Biotechnologie) Mans, R. (TU Delft BT/Industriele Microbiologie) Department BT/Biotechnologie Date 2019 Abstract Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1, which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor. Subject 5-Aminolevulinic acidCompartmentalizationLipoylationPrecursor supplyProteomicsSuccinyl-CoA To reference this document use: http://resolver.tudelft.nl/uuid:e84bed8a-7b39-4b5a-9d97-34baf03454d4 DOI https://doi.org/10.1016/j.ymben.2019.10.001 ISSN 1096-7176 Source Metabolic Engineering, 56, 190-197 Part of collection Institutional Repository Document type journal article Rights © 2019 N. Baldi, James C. Dykstra, M.A.H. Luttik, Martin Pabst, Liang Wu, Kirsten R. Benjamin, André Vente, J.T. Pronk, R. Mans Files PDF 1_s2.0_S1096717619303325_main.pdf 773.02 KB Close viewer /islandora/object/uuid:e84bed8a-7b39-4b5a-9d97-34baf03454d4/datastream/OBJ/view