An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae

Abstract

Background: Saccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis.

Results: Consistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD⁺-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose-sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high K_m for CO₂ of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO₂. Prolonged chemostat cultivation on glucose-sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway.

Conclusions: Combination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

Keywords: CO2; Electrons; Ethanol; Fermentation; NADH; Redox engineering; Sorbitol; Yeast.