Glucose feeds the TCA cycle via environmental ethanol in fermenting yeast

11 Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by 12 glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual 13 cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that 14 similar uncoupling occurs in the yeast Saccharomyces cerevisiae. Even in fermenting yeast that are net 15 releasing ethanol, media 13 C-ethanol rapid enters and is oxidized to acetaldehyde and acetyl-CoA. This is 16 evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2 H-tracing 17 reveals that ethanol is also a major source of both NADH and NADPH, and this role is augmented under 18 oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via 19 rapid reversible reactions is an ancient and conserved feature of eukaryotic metabolism.


Introduction 21
Fermentation occurs widely across Kingdoms, converting glucose into organic waste products 1-4 . In mammals, 22 the main such product is lactate. Until recently, it was commonly assumed that the liver and kidney were 23 special in their capacity to clear circulating lactate, reconverting the waste (lactate) into fuel (glucose). New 24 evidence suggests, however, that most mammalian tissues take up circulating lactate and oxidize it via the 25 TCA cycle 5 . Indeed, it seems that most carbohydrate oxidation in mammals, rather than occurring by a tissue 26 taking up glucose and fully oxidizing it to carbon dioxide, instead involves carbon flowing through circulating 27 lactate as a metabolic intermediate. Thus, glycolysis is uncoupled from the TCA cycle via cellular uptake 28 and/or excretion of lactate 5 . Biochemically, this occurs through the rapid reversible reactions linking 29 intracellular pyruvate, via lactate dehydrogenase and monocarboxylate transporters, to circulating lactate. 30 Baker's yeast (Saccharomyces cerevisiae) is a prototypical fermentative unicellular organism 6 . Its rapid 31 catabolism of glucose into ethanol + CO2 plays a central role in human society, contributing to such diverse 32 fields as baking, beverages, and biofuels. S. cerevisiae is capable of growing aerobically on substrates including galactose, glycerol, and ethanol. But when provided with ample glucose, it will ferment even in the 34 presence of adequate oxygen 7 . When glucose runs out, after a delay to rewire metabolism, aerobic growth will 35 resume (the diauxic shift) 8 . As glucose fermentation in yeast parallels aerobic glycolysis in mammals, we were 36 curious whether it similarly involves reversible excretion and uptake of the "waste product" (ethanol), rather 37 than unidirectional waste elimination. We further wondered whether any such ethanol uptake during net 38 fermentative metabolism might contribute to yeast's metabolic robustness. 39 Understanding these questions is relevant both for basic science and for bioengineering (e.g. as ethanol 40 uptake might sometimes be disadvantageous). With these motivations in mind, we show that, even when 41 fermenting, yeast actively exchanges environmental ethanol for intracellular acetaldehyde at a sufficiently rapid 42 rate that intracellular acetyl units come substantially from environmental ethanol rather than directly from 43 glucose. Moreover, such exchange enables ethanol to be a major source of NADH and NADPH, especially 44 under oxidative stress conditions. 45

Results 46
Fermenting yeast assimilate environmental ethanol 47 Ethanol can enter and exit cells via simple diffusion 9 . Thus, exogenous ethanol may enter yeast, even if they 48 are simultaneously excreting ethanol made internally from glucose. To differentiate two-carbon (2C) units from 49 environmental ethanol versus internal glucose catabolism, we grew yeasts in typical minimal media (yeast 50 nitrogen base, aerated, 30⁰C) with unlabeled glucose until mid-exponential phase. We then pelleted the cells 51 and resuspended them in yeast nitrogen base containing both glucose and ethanol, whose isotopic 52 composition is under experimental control. The glucose and ethanol concentrations in the resuspension media 53 were selected to approximate those naturally occurring during mid-exponential yeast growth in yeast nitrogen 54 base with glucose as the carbon source (recognizing that by mid-exponential phase yeast will have converted 55 a substantial amount of glucose into ethanol). Specifically, we provided glucose and ethanol at either 56 equimolar concentrations (42 mM each) or 1:1 mixture based on number of carbon atoms (28 mM glucose and 57 84 mM ethanol, "equicarbon") ( Figure 1A). 58 We used 13 C NMR to measure rates of glucose uptake (fgluc_up) and conversion to ethanol via pyruvate 59 decarboxylase (fpdc) from the cultures with [U-13 C]glucose and unlabeled ethanol ( Figure 1B). The rates 60 measured by 13 C NMR (fgluc_up and fpdc) are similar among strains of S. cerevisiae with different respiratory 61 capacity (FY4 and CEN.PK) and media substrate ratios (equimolar or equicarbon) (Supplementary figure 1A). 62 In parallel, net ethanol flux was measured by 1 H NMR, revealing active fermentation (i.e. net ethanol excretion) 63 ( Figure 1C). 64 The yeast ethanol assimilation pathway involves oxidation of ethanol to acetate, which is converted into 65 cytosolic acetyl-CoA by acetyl-CoA synthetases 10 . In attempt to directly trace potential ethanol uptake and 66 utilization, we directly measured cellular acetyl-CoA labeling distributions by LC-MS in yeasts grown with 67 unlabeled glucose and labeled ethanol ( Figure 1D), finding substantial labeling (more than 50%) from environmental ethanol ( Figure 1E). This high [M+2] labeled fraction of acetyl-CoA is consistent across strains 69 and media compositions (Supplementary Figure 1B). 70 We built a 13 C metabolic flux model to estimate the reversibility of the ethanol assimilation pathway ( Figure 1F). 71 The model was constrained by the measured glucose uptake rate, the net ethanol excretion rate, PDC flux (the 72 flux representing gross glucose conversion to ethanol), and acetyl-CoA labeling from [U-13 C]ethanol. The 73 model confirmed low PDH and high PDC flux, as typical for fermenting yeast ( Figure 1G). Notably, it revealed a 74 fast exchange flux between ethanol and acetaldehyde, with ethanol a major source of acetaldehyde even 75 though net flux is in the direction of ethanol excretion ( Figure 1G). This rapid exchange flux explains the 76 substantial acetyl-CoA labeling from environmental ethanol ( Figure 1D). To quantify fraction of cytosolic acetyl-CoA coming from environmental ethanol, we fit the observed fatty acid 84 mass isotope distribution to a binomial, reflecting the fact that each 2C unit incorporated into fat is selected 85 stochastically, with the assumption that only labeled fatty acids are newly synthesized. Such fitting revealed 86 around 60% environmental ethanol contribution ( Figure 2C). Thus, rather than being derived mainly internally 87 by glycolysis and subsequent pyruvate catabolism, when environmental ethanol is present, cytosolic acetyl-88 CoA in Baker's yeast also comes from ethanol. 89

Environmental ethanol supplies mitochondrial acetyl-CoA 90
Formation of cytosolic acetyl-CoA from acetate is catalyzed by acetyl-CoA synthetases 11,12 . Such synthetases 91 are not known in mitochondria. Accordingly, we were curious whether environmental ethanol could also 92 contribute to mitochondrial acetyl-CoA. To this end, using the same tracing strategy as above, we examined 93 whether environmental ethanol would label a metabolic product that is produced mitochondrially from acetyl - acetyl-CoA labeling zero in this deletion strain. Notably, the deletion strain nevertheless has similar whole cell 105 [M+2] acetyl-CoA labeling from ethanol (supplementary figure 4), implying that only a small fraction of total 106 cellular acetyl-CoA is mitochondrial, with Ach1 the key mitochondrial acetate assimilation enzyme. 107 Mitochondrial acetyl-CoA contributes to the TCA cycle via citrate synthase ( Figure 4A). Consistent with this, 108 from the 13 C2-ethanol tracing, we observed [M+2] (iso)citrate, aconitate, α-ketoglutarate, and succinate ( Figure  109 4B, supplementary figure 5). Fumarate, malate, and aspartate (whose carbon skeleton comes from 110 oxaloacetate) remained, however, largely unlabeled ( Figure 4C, supplementary figure 5). The extensive 111 labeling of succinate with limited labeling of fumarate or oxaloacetate pinpoints succinate dehydrogenase 112 (complex II in the electron transport chain) as being functionally blocked during yeast fermentative growth 14 . 113 Instead of being made by TCA turning, oxaloacetate and malate are generated by pyruvate carboxylase, using 114 pyruvate made from glucose. Nevertheless, acetate from environmental ethanol is assimilated into the TCA 115 cycle and drives conversion of these four-carbon TCA intermediates into citrate, α-ketoglutarate, and α-116 ketoglutarate's amino acid products. 117

Acetaldehyde oxidation feeds NADPH 118
Oxidation of ethanol to acetaldehyde generates NADH. Further oxidation of acetaldehyde into acetate via 119 aldehyde dehydrogenase generates NADPH. When the canonical main NAPDH-production pathway, the 120 oxidative pentose phosphate pathway, is deleted, the acetaldehyde dehydrogenase Ald6 is essential for yeast 121 growth 15 . We were curious about the extent to which acetaldehyde derived from environmental ethanol 122 contributes to NADH or NADPH. To explore this, we transferred fermenting yeast into glucose:ethanol as 123 above, with either the glucose or the ethanol deuterium labeled. Specifically, we compared NADH and NADPH 124 labeling from [1-2 H]glucose (the labeled hydrogen is transferred to NADPH via G6PD, encoded by gene zwf1) 125 and [1,1-2 H2]ethanol (the labeled hydrogen is transferred to NADH by ADH and to NADPH via Ald) ( Figure  126 5A,D). Direct measurement of 2 H-labeling in NADH and NADPH is technically challenging due to limited 127 abundance and stability, but nevertheless we observed clear isotope shifts upon exposure to the 2 H-labeled 128 substrates, confirming contribution from glucose's position 1 hydrogen to NADPH and from ethanol's position 1 129 hydrogens to both NADH and NADPH (Supplementary Figure 5B,C,E,F). 130 To obtain more precise and compartment-specific information, we used fatty acid labeling to read out cytosolic 131 NADPH labeling. Fatty acid synthesis incorporates 2 NADPH hydrides per acetyl group ( Figure 5A) 16 . 132 Strikingly, we observed greater deuterium labeling of fatty acids from [1,1-2 H2]ethanol than from [1-2 H]glucose 133 (Supplementary figure 7,8). This reflects a major contribution of ethanol-derived acetaldehyde to cytosolic 134 NADPH via Ald6 ( Figure 5B). 135 To convert the observed labeling into quantitative contributions to NADPH, we need to account for deuterium 136 loss from NADPH via hydrogen-deuterium exchange with water mediated by flavin enzymes 16 figure 6G,H,I, 9). Correcting for such exchange (and for extent of substrate labeling), we 141 observed that the oxPPP and Ald6 together account for most cytosolic NADPH, with the contribution of ethanol 142 via Ald6 roughly double that of glucose via the oxidative pentose phosphate pathway ( Figure 5D). 143 Consistent with ethanol oxidation and oxPPP being alternative cytosolic NADPH production pathways, in 144 ∆ald6, oxPPP contribution to NADPH production (based on fatty acid labeling patterns) is nearly twice as high 145 as in wild type ( Figure 5C, supplementary figure 7). In ∆zwf1, Ald6 contribution to NADPH production (based 146 on fatty acid labeling patterns) similarly doubles ( Figure 5B, supplementary figure 8). Thus, ethanol is an 147 important source of both acetyl and hydride units in Baker's yeast. 148

Ethanol becomes a yet greater NADH and NADPH source in response to peroxide stress 149
We were curious whether cells might shift between glucose or ethanol as an NAD(P)H source in response to 150 environmental conditions. To explore this possibility, we grew yeast in glucose:ethanol with one substrate 2 H-151 labeled as above, spiked in H2O2 to a final concentration of 20 mM, and rapidly sampled metabolites and their 152 labeling 18,19 ( Figure 6A). Upon adding H2O2, the NADH concentration and NADH/NAD + ratio fell markedly 153 ( Figure 6B, C). Such a drop was expected, given that oxidative stress is known oxidize the GADPH's active 154 site cysteine and thereby block glycolytic flux and NADH production. Consistent with GAPDH being shut off, in 155 addition to an increase of fructose-1,6-bisphosphate (FBP) (Supplementary Figure 10), we observed increased 156 NADH labeling from ethanol, which became the dominant NADH hydride source ( Figure 6D). Thus, ethanol 157 catabolism is a crucial source for NADH when glycolysis is blocked by oxidative stress. 158 A classical rationale for glycolytic blockade by oxidative stress is to divert flux into the oxidative pentose 159 phosphate pathway to help maintain NADPH homeostasis. Notably, the same concentration of hydrogen 160 peroxide that markedly suppressed NADH had no overt effect on NADPH pool size or the NADPH: NADP + ratio 161 ( Figure 6E, F). However, rather than increasing the oxidative pentose phosphate pathway contribution to 162 NADPH as measured by [1-2 H]glucose, this contribution was decreased, with ethanol's contribution to NADPH 163 markedly increased ( Figure 6G). Thus, in contrast to the common assumption that the main NADPH production 164 route during oxidative stress is the oxidative pentose phosphate pathway, we observe an acute shift towards a 165 greater NADPH contribution from ethanol oxidation under H2O2 stress 15,20 . 166

Discussion 167
A fundamental metabolic question is, "Which pathways are coupled versus independent?" Here we present 168 evidence that uncoupling of glycolysis from the TCA cycle is an evolutionarily conserved design principle in 169 eukaryotic metabolism. Specifically, we show that fermenting Baker's yeast simultaneously release and uptake 170 ethanol, much as many mammalian cells simultaneously produce and consume circulating lactate. Both lactate 171 and ethanol are redox-balanced with glucose 21 . Thus, their release allows glycolysis to run without need for the 172 TCA cycle or oxidative phosphorylation. Release of these electron-rich products anticorrelates with internal 173 NADH consumption by the electron transport chain 22 .
While the net release of ethanol by fermenting yeast has been long appreciated, we are unaware of prior 175 demonstration that fermenting yeast simultaneously engage in extensive ethanol uptake. Through experiments 176 with 13 C-ethanol, we show that, under typical mid-exponential fermentative growth conditions, environmental 177 ethanol, rather than mitochondrial pyruvate catabolism, supplies a majority of both cytoso lic and mitochondrial 178 acetyl-CoA. In mitochondria, we prove that the ethanol assimilation pathway involves the CoA-transferase 179 Ach1 23-25 . The assimilated ethanol was originally produced from glucose. But the pathway from glycolysis to  Yeast strains. S. cerevisiae strain FY4, derived from S288c, was taken from in-house frozen stocks. S. 211 cerevisiae strain CEN.PK was obtained from José Avalos. S. cerevisiae prototrophic mutant strains were 212 obtained from David Botstein, which were also derived from S288c through a diploid intermediate strain 26,27 . 213 Yeast batch cell culture growth. S. cerevisiae colony was inoculated into an overnight culture, containing 6.7 214 g/L of Yeast Nitrogen Base (YNB) without amino acids and 20 g/L of glucose. After 24 h of growth at 30 °C, the 215 overnight culture was diluted 1:100 into appropriate experimental media, containing 6.7 g/L YNB without amino 216 acids and a carbon source/isotope tracer as specified for each experiment. For the prototrophic ∆zwf1 strain, 217 20 mg/L methionine was added to the media to accelerate growth. 218 1 H qNMR measurement. Yeast were first grown to mid-exponential phase (OD=0.5), split into two equal 219 portions, and centrifuged to pellets. In the equimolar case, each pellet was redissolved at equal concentration 220 ppm (dd, 1H) for β-glucose, and 1.17 ppm (t, 3H) for ethanol. Water suppression is achieved by O1P=4.66 228 ppm, spw1=0.002. To achieve quantitative NMR, D1 is set to 5 s and 90˚ pulse (p1)to 11.69 μs. 229 13 C qNMR measurement. The sample collection procedure and preparation were identical to the 1 H NMR 230 excretion profile, except that 13 C sodium formate (0.1 M) was utilized as a standard instead of deuterated TMS.

231
13C NMR δ: 168 ppm (s, 1C) for the sodium formate standard, 161 ppm (d, 1C) for glucose, and 57 ppm (d, 232 1C) for ethanol. Samples of media that the cells are switched into are measured as baseline . We observed that 233 unlabeled ethanol present in the media also gives a detectable 13 C NMR signal (s, 1C at 57 ppm) due to 234 natural 13 C abundance. This natural abundance signal was not corrected for because it is < 5% signal of [U-  Isotope tracing experiments. Cells were grown in YNB + 10g/L glucose up to OD=0.5. Then, the cells were 261 quickly centrifuged, the supernatant discarded, and switched to equimolar or equicarbon media with either 262 glucose or ethanol 13 C-or 2 H-labeled, or with both carbon sources unlabeled in 50% 2 H2O. The cells were 263 allowed to grow in the labeled media for 1 hour before harvesting. A potential concern these experiments is 264 dilution of labeled ethanol tracer by unlabeled ethanol made from glucose. As no more than 2 mM unlabeled 265 ethanol is excreted into the media during the one-hour incubation, environmental ethanol remains more than 266 95% fully labeled during the experiments. OD was taken before and after the incubation period to assure the 267 cells were in exponential growth phase during the experiment. was used for the NMR data processing. Statistical analysis are performed with GraphPad Prism, including two-294 tailed t-test (with false discovery rate correction by the two-stage step up Benjamini, Krieger, and Yekutieli 295 method to confirm that any reported significant results involving statistical comparisons of multiple isotopic 296 forms of the same metabolite remain significant after correction for the multiple comparisons ); ordinary one-297 way ANOVA (when row matching is statistically significant, RM one-way ANOVA instead); and linear trend 298 (between column mean and by left-to-right column order, with p-value from F test). For routine data 299 visualization and analysis, MATLAB 34 , R Studio, Python, and Microsoft Excel were utilized. Schematics and 300 diagrams were created with the aid of GraphPad Prism, ChemDraw, and BioRender. 301 A. When fed with labeled ethanol, the resulting 13 C-labeled acetyl-CoA is incorporated into newly 419 synthesized fatty acids. As both labeled and unlabeled cytosolic acetyl-CoA are randomly incorporated 420 into growing fatty acid chains, the resulting fatty acid mass isotope distribution follows a binomial 421 probability distribution. 422 B. Fatty acid (palmitate) labeling pattern from equimolar glucose: 13 C-ethanol co-feeding experiment as in 423 Figure 1D (mean, SE, n=3 (biological replicates)). Briefly, newly synthesized fatty acids are getting 424 labeled by [M+2] acetyl-CoA, which is a result of uptaking 13 C-ethanol from growth media by S. 425 cerevisiae. 426 C. Cytosolic acetyl-CoA labeling fitted from fatty acid labeling from equimolar glucose: 13 C-ethanol co-427 feeding experiment as in Figure 1D (mean, SE, n=6 (3 biological replicates with results from both 428 C16:0 and C18:0)). 429 B. Glutamate (Glu) and NAG labeling from the glucose: 13 C-ethanol co-feeding experiment as in Figure  436 1D, including also data for ∆ach1 yeast (thereby identifying Ach1 as an enzyme essential for 437 mitochondrial assimilation of ethanol-derived carbon into acetyl-CoA) (mean, SE, n=3 (biological 438 replicates); ***, p <.001 by t-test  A. Schematic of TCA cycle highlighting observed TCA labeling from glucose: 13 C-ethanol co-feeding as in 446 Figure 1D, where [M+2] acetyl-CoA is a result of uptaking 13 C-ethanol from growth media by S. 447 cerevisiae (Created with Biorender). 13 C is in green while 12 C is in white.

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C. Labeling patterns of pyruvate and TCA intermediates on "left hand side" of TCA cycle (with aspartate as 451 a surrogate for oxaloacetate) (mean, SE, n=3 (biological replicates)). These metabolites are not labeled 452 and thus originate exclusively from glucose, suggesting blockade of complex II and production of 453 malate/fumarate through oxaloacetate reduction. 454