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 Km 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.

Application of cofactors in the regulation of microbial metabolism: A state of the art review

“… Deng et al (2022) optimized a cascade system by regulating the intensity of NADPH gene expression to produce a higher (S)-equol titer (3418.5 mg/L) with a conversion rate of approximately 85.9%. van Aalst et al (2022) functional expression of the gene in anaerobic, slow-growing chemostat cultivation on glucose-sorbitol mixtures resulted in a 12-fold higher co-consumption rate of sorbitol than that observed in sorbitol-consuming reference strains. Rami Reddy Tadi et al (2021) metabolic engineering of Bacillus megaterium through co-expression of the precursor ( phbRBC ) and NADPH cofactor regeneration ( zwf ) genes resulted in a 2.67-fold increase in PHB accumulation.…”

Uncovering Challenges for Complete Carbohydrate to Bioethanol Utilization in a Reductive Catalytic Fractionation Biorefinery

“…This phenomenon has been reported in genetically modified S. cerevisiae strains capable of fermenting sorbitol under anaerobic conditions . While analogous strategies for xylitol are not (yet) documented, they could potentially favor the strategy of solubilizing xylose and reducing it to xylitol during RCF, given the higher theoretical ethanol yields.…”

Co-cultivation ofSaccharomyces cerevisiaestrains combines advantages of different metabolic engineering strategies for improved ethanol yield

“…In industrial ethanol fermentation, the specific growth rate decreases as the ethanol concentration reaches inhibitory levels and/or non-sugar nutrients are depleted (16). A recent study showed that, in slow-growing anaerobic chemostat cultures (0.05 h -1 ), a PRK-RuBisCO strain produced up to 80-fold more acetaldehyde and 30-fold more acetate than a reference strain (17). This production of acetaldehyde and acetate was attributed to an in vivo overcapacity of the key enzymes of the PRK-RuBisCO bypass.…”

“…This production of acetaldehyde and acetate was attributed to an in vivo overcapacity of the key enzymes of the PRK-RuBisCO bypass. Reduction of the copy number of the expression cassette for RuBisCO led to lower acetaldehyde and acetate production in slow-growing cultures and a corresponding increase in ethanol yield (17). The production of acetaldehyde and acetate was further decreased by reducing PRK activity by lowering protein abundance by a C-terminal extension of PRK, or by expressing the spinach prk gene from the growth-rate-dependent ANB1 promoter (17,18).…”

“…Reduction of the copy number of the expression cassette for RuBisCO led to lower acetaldehyde and acetate production in slow-growing cultures and a corresponding increase in ethanol yield (17). The production of acetaldehyde and acetate was further decreased by reducing PRK activity by lowering protein abundance by a C-terminal extension of PRK, or by expressing the spinach prk gene from the growth-rate-dependent ANB1 promoter (17,18). However, the resulting strains still showed trade-offs in performance at low and high specific growth rates, which illustrated the challenges involved in tuning the activity of engineered pathways under dynamic conditions.…”