Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production

Valk, Sophie Claire de; Gulik, Walter van; Jansen, Mickel L.A.; Pronk, Jack T.; Mans, Robert

doi:10.1016/j.synbio.2021.12.010

Cited by 26 publications

(21 citation statements)

References 134 publications

(180 reference statements)

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“…Compared to alcoholic fermentation of glucose, this pathway for sorbitol fermentation would yield 42% less ATP per mole of substrate. Provided that sufficient rates of alcoholic fermentation can be achieved to maintain industrially relevant productivities, a low ATP yield on sorbitol could be interesting as it should divert carbon substrate from biomass formation to ethanol production [ 27 , 28 ].…”

Section: Resultsmentioning

confidence: 99%

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

Aalst

Mans

Pronk

2022

Biotechnol Biofuels

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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.

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Section: Resultsmentioning

confidence: 99%

“…To further assess the predicted impact of the proposed metabolic engineering strategy, it was implemented in a stoichiometric model of the core metabolic network of S. cerevisiae [ 27 , 29 ]. The model was then used to calculate biomass and ethanol yields at different specific growth rates.…”

Section: Resultsmentioning

confidence: 99%

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

Aalst

Mans

Pronk

2022

Biotechnol Biofuels

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“…Additionally, for industrial implementation, engineered yeast cells should be able to cope with high maintenance energy requirements that arise due to such high ethanol concentrations and the presence of other inhibitors in the culture [ 43 – 46 ]. Therefore, in addition to other practical considerations for efficient use of such engineered strains in industrial fermentations [ 47 ], the expressed sugar transporters should allow for very high dissimilation rate for maintenance energy provision.…”

Section: Discussionmentioning

confidence: 99%

Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield

Valk

Bouwmeester

Hulster

et al. 2022

Biotechnol Biofuels

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Background In the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose–proton symport. Results Introduction of heterologous glucose– or fructose–proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose–proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose−1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose–proton symporter, fructose–proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent−1) and 46.6% decreased biomass yield on sucrose. Conclusions This study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.

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“…Ethanol is predominantly used as a renewable ‘drop-in’ transportation fuel and a feedstock for production of other compounds. van Aalst et al reviewed pathway engineering strategies for improving ethanol yield of anaerobic fermentation of sugars [ 9 ]. For heterologous production of spinosad in Streptomyces albus , An et al engineered the polyketide skeleton and precursor supply, which resulted the highest spinosad titer of 70 mg/L in a heterologous Streptomyces species [ 10 ].…”

Section: Pathway Engineeringmentioning

confidence: 99%

Pathway and protein engineering for biosynthesis

Zhou

Grininger

Alper

2022

Synthetic and Systems Biotechnology

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Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production

Cited by 26 publications

References 134 publications

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

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

Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield

Pathway and protein engineering for biosynthesis

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