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

BackgroundAnaerobic 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.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-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 x 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 x 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 x cbbm strain. At 0.05 h(-1), this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 x 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.ConclusionsAcetaldehyde 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.

Automated summary

In slow-growing anaerobic cultures, it was found that the introduction of Calvin-cycle enzymes in Saccharomyces cerevisiae strain can improve ethanol production but also lead to the formation of excessive acetaldehyde and acetate. By reducing the expression level of RuBisCO and PRK and using a growth rate-dependent promoter, the production of acetaldehyde and acetate can be reduced and glycerol production efficiency can be increased.