title: The bacterial Entner-Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron-sulfur cluster enzyme 6-phosphogluconate dehydratase.
abstract: Replacement of the glycolytic pathway of Saccharomyces cerevisiae by a bacterial Entner-Doudoroff pathway (EDP) would result in lower ATP production and therefore a lower biomass yield is expected that would further allow higher products yields in the fermentation of sugars. To establish catabolism of glucose via the EDP in S. cerevisiae requires expression of only two additional enzyme activities, 6-phosphogluconate dehydratase (PGDH) and KDPG aldolase. In this work, KDPG aldolase from Escherichia coli could be successfully expressed in the yeast cytosol with very high enzyme activity. Nevertheless, simultaneous expression of KDPG aldolase and a codon optimized PGDH gene of E. coli could not replace glycolysis or the pentose phosphate pathway in growth experiments. It could be shown that this was due to the very low enzyme activity of PGDH. This bacterial enzyme is a [4Fe-4S] iron-sulfur cluster protein. Several attempts to improve the availability of iron-sulfur clusters or iron in the yeast cells, to attract the iron-sulfur cluster assembly machinery to Leu1-PGDH fusion proteins or to localize the PGDH in the mitochondria did not result in improved enzyme activities. From our results we conclude that establishing functional expression of iron-sulfur cluster enzymes will be a major task for the integration of the EDP and other biochemical pathways in yeast.
authors: Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany.
authors: Feline
authors: F
authors: Benisch
authors: Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany. Electronic address: e.boles@bio.uni-frankfurt.de.
authors: Eckhard
authors: E
authors: Boles
mesh_terms: D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis / Q000235:genetics*; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D003062:Codon; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D006836:Hydro-Lyases; C022362:phosphogluconate dehydratase
keywords: Entner–Doudoroff pathway; Iron–sulfur cluster; KDPG aldolase; Metabolic engineering; Product yield; Saccharomyces cerevisiae