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Background  Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast  Saccharomyces cerevisiae . Anaerobic cultures of wild-type  S. cerevisiae  require formation of glycerol to maintain the intracellular NADH/NAD +  balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in  S. cerevisiae  was shown to enable reoxidation of NADH with CO 2  as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.    Results  Multi-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the  Escherichia coli  GroEL/GroES chaperones and expression of PRK from the anaerobically inducible  DAN1  promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting  S. cerevisiae  strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of  GPD2 , which encodes an isoenzyme of NAD + -dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO 2 -reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.    Conclusions  The metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of  S. cerevisiae , with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.    Electronic supplementary material  The online version of this article (10.1186/s13068-017-1001-z) contains supplementary material, which is available to authorized users.

biotechnology for biofuels

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield