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

Abstract

BackgroundReduction 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 CO2 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.ResultsMulti-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 CO2-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.ConclusionsThe 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 materialThe online version of this article (10.1186/s13068-017-1001-z) contains supplementary material, which is available to authorized users.

Synthetic Biology Applied to Carbon Conservative and Carbon Dioxide Recycling Pathways

“…In accordance with this idea, overexpression of non-oxidative PPP genes RPEI, TKL1, TKL2, TAL1, RKI1 in the engineered strain also deleted for GPD2 resulted in a specific growth rate that was virtually the same as the reference strain under anaerobic batch condition on glucose. Furthermore, this engineered strain achieved the best fermentation performance with an increased ethanol yield of 15% and a 90% decrease of glycerol production (Papapetridis et al, 2018).…”

“…In their design, Ru5P must be produced from the non-oxidative pentose phosphate route in order to reoxidize all biosynthetic NADH and not NADPH through a transhydrogenase-type conversion NADPH-> NADH (Guadalupe-Medina et al, 2013). This clever idea was demonstrated in two elegant papers published in Biotechnology for Biofuels (Guadalupe-Medina et al, 2013;Papapetridis et al, 2018). In the first paper, the authors expressed the codon-optimized prokaryotic form II RuBisCO-encoding cbbM gene from T. denitrificans in a centromeric plasmid under the strong TDH3 promoter and showed that RuBisCO was functional only upon co-expression of E. coli groEL/groES encoding chaperones whereas surprisingly T denitrificans chaperones encoded by cbb02/cbbQ2 were ineffective.…”

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

“…(3) Forced co-consumption in which simultaneous utilization of D-xylose and D-glucose was achieved by deleting the genes encoding glucose-6-phosphate isomerase (Pgi1) and ribulose-5-phosphate epimerase (Rpe1) in a xylose-isomerasebased xylose-fermenting strain. Evolutionary engineering yielded mutations in Hxk2 which improved the rate of the forced coconsumption of D-xylose and D-glucose (Papapetridis et al, 2018). The latter strategy, however, requires stoichiometric mixtures of these two sugars.…”

Evaluation of Rice Bran as a Supplement for Production of Bioethanol by Saccharomyces cerevisiae

“…The kinetic study of the process correlates the formation of the product of interest with the nutrients consumption. Since ethanol formation cannot occur without the presence of cells, it is expected that the microorganism growth and the product formation are closely related (Papapetridis et al, 2018). Among the most used mathematical models is the equation developed by Monod (1949), which describes the effect of limiting growth as a function of the specific growth rate.…”