Quantification and mitigation of byproduct formation by low-glycerol-producing Saccharomyces cerevisiae strains containing Calvin-cycle enzymes

A.C.A. van Aalst, Mickel L.A. Jansen, R. Mans, J.T. Pronk

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Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results: In slow-growing anaerobic chemostat cultures (D = 0.05 h −1), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h −1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h −1) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h −1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower. Conclusions: Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.

Original languageEnglish
Article number81
Number of pages17
JournalBiotechnology for Biofuels and Bioproducts
Issue number1
Publication statusPublished - 2023


  • Chemostat
  • Acetaldehyde
  • Acetate
  • Phosphoribulokinase
  • RuBisCO
  • Anaerobic
  • Ethanol
  • Redox


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