BackgroundAnaerobicSaccharomyces cerevisiaecultures 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.ResultsIn 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-encodingcbbmexpression 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 ×cbbmstrain. These modifications did not affect glycerol production at 0.05 h−1but 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 ×cbbmstrain. In another strategy, the promoter ofANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 ×cbbmstrain. At 0.05 h−1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 ×cbbmstrain, 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.ConclusionsAcetaldehyde and acetate formation by slow-growing cultures of engineeredS. cerevisiaestrains 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.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02329-9.