Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products
Journal article, 2020

Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only.

Mathematical modelling

Saccharomyces cerevisiae

Synthetic biology

Non-oxidative glycolysis

3-Hydroxypropionic acid

Metabolic engineering


John Hellgren

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Novo Nordisk Foundation

Alexei Godina

Total S.A.

Jens B Nielsen

BioInnovation Institute

Novo Nordisk Foundation

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Verena Siewers

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Novo Nordisk Foundation

Metabolic Engineering

1096-7176 (ISSN) 1096-7184 (eISSN)

Vol. 62 150-160

Subject Categories

Other Basic Medicine


Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)





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