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Glucose feeds the tricarboxylic acid cycle via excreted ethanol in fermenting yeast

Abstract

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis. Even in fermenting S. cerevisiae that is net releasing ethanol, media 13C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.

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Fig. 1: Environmental ethanol is a major source of acetyl units in fermenting S. cerevisiae.
Fig. 2: Environmental ethanol contributes to both cytosolic and mitochondrial acetyl-CoA in fermenting S. cerevisae.
Fig. 3: Carbons from ethanol feed into TCA intermediates.
Fig. 4: Ald6 is a major source of the cytosolic NADPH.
Fig. 5: Instead of oxPPP, ethanol oxidation provides major fraction of active hydrides in the presence of H2O2.

Data availability

Data are shown in the figures and supplementary figures. Requests for materials can be made to the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank S. Silverman and D. Botstein for access to the yeast knockout collection; T. TeSlaa, R. Ryseck and A. J. Cowan for feedback on the manuscript; I. Pelczer and J. Eng for assistance with NMR and mass spectrometry; C.M. Call for help with preliminary experiments; and M. Seyedsayamdost and members of the Rabinowitz laboratory for helpful discussions. Services, results and/or products in support of the research project were generated by the Rutgers Cancer Institute of New Jersey Metabolomics Shared Resource, supported, in part, with funding from NCI-CCSG P30CA072720-5923. This work was funded by U.S. Department of Energy grant DE-SC0018260 and the Department of Energy Center for Advanced Bioenergy and Bioproducts Innovation (award no. DE-SC0018420). Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy.

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Authors and Affiliations

Authors

Contributions

T.X. and J.D.R. designed the study. T.X. performed NMR studies. T.X. and A.K. performed isotopic tracing studies. T.X., Y.H., L.C. and A.K. developed the computational models. T.X. and A.K. analyzed the data. J.D.R. and T.X. wrote the manuscript, with the help from all authors.

Corresponding author

Correspondence to Joshua D. Rabinowitz.

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Nature Chemical Biology thanks Jens Nielsen, Kiran Patil and Aljoscha Wahl for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Environmental ethanol consistently provides acetyl units across two S. cerevisiae strains.

(a) Ethanol production rate from [U-13C]glucose (mean, SE, n=3 biological replicates). Results are similar to literature29 value of 10.5 mmol*(OD*h)−1. (b) Glucose uptake rate from the same experiment as in a (mean, SE, n=3 biological replicates), comparable to literature value29 of 6.9-7.6 mmol*(OD*h)−1. (c) The [M+2] labeling of acetyl-CoA in S.cerevisiae FY4 with equimolar glucose and [U-13C]ethanol measured as a function of time in labeled ethanol (mean, SE, n=3 biological replicates). The consistent labeling fraction from 30 min to 1 h implies that 1 h is a pseudo-steady-state measurement. (d) After incubation with glucose and [U-13C]ethanol for 1 hour at varying conditions and strains, the [M+2] labeled acetyl-CoA fraction from the cell (mixture of cytosolic and mitochondrial origins) was directly measured by LC-MS (mean, SE, n=3 biological replicates). (e) Examples of q13C NMR spectra of the yeast culture media upon addition of [U-13C]glucose and after S. cerevisiae growth for 1 h.

Source data

Extended Data Fig. 2 Environmental ethanol does not enter glycolytic intermediates in fermenting S. cerevisiae.

(a) After natural isotope correction, no meaningful [M+2] fraction is observed in glycolytic intermediates: fructose-1,6-phosphate (FBP), dihydroxyacetone phosphate (DHAP), hexose-6-phosphates (G6P+F6P), or UDP-D-glucose (S. cerevisiae, equimolar glucose:[U-13C]ethanol, mean, SE, n=3 biological replicates). (b) As in a, for equicarbon condition. (c) Example of the natural abundance observed in raw mass spectra of FBP. The natural abundance is corrected by the binomial distribution model37 to arrive at the labeling patterns reported throughout the manuscript including a and b above.

Source data

Extended Data Fig. 3 Environmental ethanol feeds fatty acid synthesis to a similar extent across the equimolar and equicarbon conditions.

(a) 13C-isotope labeling pattern of palmitic acid from S. cerevisiae FY4 (S288c) switched to and incubated in minimal media (YNB) with equimolar or equicarbon glucose: [U-13C]ethanol (mean, SE, n=3 biological replicates). (b) 13C isotope labeling pattern of stearic acid from the same experiments as shown in a (mean, SE, n=3 biological replicates). (c) Whole-cell and cytosolic acetyl-CoA labeling from [U-13C]ethanol is similar. Whole-cell labeling is directly measured by LC-MS of acetyl-CoA (mean, SE, n=3 biological replicates). Cytosolic labeling is inferred from fatty acids labeling patterns (mean ± SE, results from both C16:0 and C18:0 are averaged, resulting a total n=6 measurements from n=3 biological replicates). The directly measured whole-cell acetyl-CoA is the same data as shown in Fig. 1e.

Source data

Extended Data Fig. 4 Ethanol contribution to mitochondrial acetyl-CoA is blocked by knocking out ACH1.

(a) Glu and NAG labeling from the experiment in Fig. 1d with equicarbon glucose: [U-13C]ethanol (mean, SE, n=3 biological replicates; ***, p<.001, by two-sided t-test). Briefly, while Glu is labeled up to [M+2], NAG [M+4] arises from the reaction of [M+2] Glu with mitochondrial [M+2] acetyl-CoA, which depends on Ach1. (b) Mitochondrial acetyl-CoA [M+2] fraction fitted from glutamate and NAG labeling in a (mean, SE, n=3 biological replicates). (c) Schematic showing ACH1 as the exclusive point of entry for carbons from ethanol or acetate into mitochondrial acetyl-CoA (Created with BioRender). (d) Directly measured cellular acetyl-CoA [M+2] labeled fraction is similar across media conditions or strains including the ACH1 knockout strain (mean, SE, RM one-way ANOVA with Geisser-Greenhouse correction, p= .097 (ns), .19(ns), n=3 biological replicates). FY4 is isogenic to S288c and ∆ach1 is from S288c, while CEN.PK is derived from ENY.WA-1A and MC996A.

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Extended Data Fig. 5 Carbons from [U-13C]ethanol feed into TCA intermediates across media conditions and budding yeast strains/species.

Some data for S.c. FY4 and I.o. SD108 are repeated from main Fig. 3b,c (mean, SE, n=3 biological replicates).

Source data

Extended Data Fig. 6 The environmental ethanol contribution to TCA intermediates is concentration-dependent.

Labeling pattern of TCA intermediates from the indicated budding yeast grown starting at OD = 0.1 in standard high glucose media (55 mM regular glucose) with the indicated concentrations of [U-13C]ethanol for 1 h (mean, SE, n=3 biological replicates). Note that, for the lower 13C-ethanol concentrations, the labeled ethanol is substantially diluted by unlabeled ethanol made from the unlabeled glucose during the duration of the experiment. In S. cerevisiae, this is about a 10-fold dilution for the 50 μM condition and 2-fold for the 500 μM condition. Thus, the above labeling patterns conservatively underestimate the contribution of low concentrations of environmental ethanol to TCA intermediates.

Source data

Extended Data Fig. 7 Deuterium tracing into NAD(P)H is similar across media conditions and strains.

(a) Scheme depicting NADP2H and NAD2H production from [1,1-2H2]ethanol isotope tracer. (b) Labeled fractions of NADPH active hydride with the tracer in a. The values are computed from matrix decompositions of labeling distributions of NAD(P)H and NAD(P)+ that are directly measured by LC-MS (mean, SE, n=3 biological replicates). (c) As in b, for NADH (mean, SE, n=3 biological replicates). (d) Chemical scheme depicting NADP2H production from [1-2H]glucose via the first step of oxPPP. (e) Labeled fractions of NADPH active hydride with the tracer in d (mean, SE, n=3 biological replicates). (f) As in e, for NADH (mean, SE, n=3 biological replicates). (g) Chemical basis of D2O active hydride exchange with NAD(P)H16. (h) The fraction of NADPH hydride exchanged with water, as measured in media swap experiment with 50% D2O (mean, SE, n=3 biological replicates). (i) As in h, for NADH (mean, SE, n=3 biological replicates).

Source data

Extended Data Fig. 8 ALD6 knockout shifts NADPH production (and thus fatty acid hydride labeling) towards the oxidative pentose phosphate pathway and ZWF knockout shifts it towards the ethanol-ALD6 pathway.

(a) 2H isotope labeling pattern of palmitic and stearic acids from S. cerevisiae FY4 and ∆ald6 swapped into equimolar [1-2H]glucose: ethanol for 1 h (mean, SE, n=3 biological replicates). (b) As in a, for equicarbon [1-2H]glucose: ethanol (mean, SEM, n=3 biological replicates). (c) 2H isotope labeling pattern of palmitic and stearic acids from S. cerevisiae FY4 and ∆ald6 swapped into equimolar glucose: [1,1-2H2]ethanol for 1 h (mean, SE, n=3 biological replicates). (d) As in c, for equicarbon glucose: [1,1-2H2]ethanol (mean, SE, n=3 (biological replicates)).

Source data

Extended Data Fig. 9 Fatty acids in S. cerevisiae are labeled from D2O (50%) reflecting direct water incorporation during fatty acid synthesis and H-D exchange between water and NADPH.

(a) 2H isotope labeling pattern of palmitic acid from S. cerevisiae FY4 swapped into regular glucose media with 50% D2O for 1 h (mean, SE, n=3 biological replicates). (b) As in a, for stearic acid.

Source data

Extended Data Fig. 10 The fructose-1,6-bisphosphate (FBP) pool size acutely increases in response to oxidative stress.

FBP pool size, after treatment with 20 mM hydrogen peroxide in S. cerevisiae FY4 grown in glucose+ethanol. The increase in FBP pool size after the oxidative stress is statistically significant (mean, SE, n=6 biological replicates, slope > 0 by linear regression, p=0.03).

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Xiao, T., Khan, A., Shen, Y. et al. Glucose feeds the tricarboxylic acid cycle via excreted ethanol in fermenting yeast. Nat Chem Biol 18, 1380–1387 (2022). https://doi.org/10.1038/s41589-022-01091-7

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