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

• Bacterial KDPG aldolase is expressed with high activity in Saccharomyces cerevisiae.
• Codon optimization of 6-phosphogluconate dehydratase (PGDH) from Escherichia coli results in high expression of this enzyme in yeast.
• Activity of PGDH and therefore of Entner–Doudoroff pathway (EDP) in Saccharomyces cerevisiae was very low.
• It is suspected that insufficient expression or assembly of iron–sulfur cluster limits the activity of PGDH.
• Establishing functional expression of iron–sulfur cluster enzymes will be a major task for the integration of the EDP and other synthetic pathways in yeast.

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.