Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism

Abstract

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults^1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p^3-5 in maintaining oxidant resistance is unknown⁶. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.

Extended data Figure 1. Proteomic analysis.

A proteomic time-course experiment recorded by SWATH-MS , was re-analysed with a new spectral library and a recent version of Spectronaut (Biognosys) software, increasing the depth of the proteomic analysis. a, Principal component analysis shows that proteome profiles of H₂O₂-exposed Δtpo1 yeast are more different from the wild type than that of a Tpo1p-overexpressing strain and that there is a metabolic adaptation in the lysine pathway. Numbers indicate time points (in minutes) after the oxidative insult. b, Gene Ontology (GO) enrichment analysis shows that a number of biosynthetic processes are significantly enriched in proteins differentially expressed between wild-type and Δtpo1 yeast upon exposure to H₂O₂. Most of the GO terms that are significantly enriched belong to processes that are typically seen to be activated upon treatment with H₂O₂, including oxidation-reduction process, cell cycle, nucleotide synthesis and ribosome . Metabolic processes affected include carbohydrate- and amino acid metabolism, including two GO terms specific for the lysine pathway. Significance of enrichment over base frequency was calculated using a binominal test , and P values (red line) were corrected for false discovery rate using Bonferroni (red) or Benjamini-Hochberg (green) correction. n = 5.

Extended data Figure 2. The S. cerevisiae lysine biosynthesis pathway via aminoadipate.

Lys20p/Lys21p, homocitrate synthase; Aco2p, aconitase; Lys4p, homoaconitase; Lys12p, homo-isocitrate dehydrogenase; Aro8p, 2-aminoadipate transaminase; Lys2p, α-aminoadipate reductase; Lys9p, saccharopine dehydrogenase (NADP⁺ and L-glutamate forming); Lys1p, saccharopine dehydrogenase (NAD⁺ and L-lysine forming).

Extended data Figure 3. Effect of lysine supplementation on the intracellular concentration of the canonical polyamines and growth.

a, Polyamine content in yeast cells in the mid-log phase grown in SM media with and without 25 μg ml^-1 of lysine, as quantified by LC-MS/MS using selected reaction monitoring. Bar charts represent the mean ± s.d.; n = 3 biologically independent samples for non-supplemented conditions and n=4 for supplemented conditions. Unpaired two-tailed Student’s t-test, wild-type cells supplemented versus non-supplemented with lysine: **** P ≤ 0.0001, ** P = 0.0066. b, Growth curves of wild-type cells grown in SM media with or without lysine (25 μg ml^-1) supplementation. The data represent mean ± s.d.; n = 3 biologically independent samples.

Extended data Figure 4. Predictions of the growth-optimized (naive) model for flux balance analysis of lysine harvesting.

We used a revised version of the S. cerevisiae genome-scale metabolic model (iMM904_NADcorrected ). Six additional reactions related to cadaverine biosynthesis were added: two metabolic reactions for ornithine decarboxylase (H + lys_L --> CO₂ + cadaverine) and spermine synthase (S-adenosyl 3-(methylthio) propylamine + cadaverine --> S-methyl-5'-thioadenosine + aminopropyl-cadaverine), and four reactions for transporting and exchanging cadaverine and aminopropylcadaverine. In silico synthetic minimal media constraints were used according to the original model (iMM904 ). For excess lysine uptake, the constraint of L-lysine exchange reaction was fixed to value of 1. Model simulation (FBA) was performed using the COBRA toolbox for maximum growth in both media conditions.

Extended data Figure 5. Lysine and methionine supplementation have no growth-relevant effect on the oxidative capacity of media containing H₂O₂.

a, H₂O₂ levels measured by Amplex Red fluorescence in water (left) or SM medium (right) supplemented with L-lysine (25 μg ml^-1), D-lysine (25 μg ml^-1) or L-methionine (40 μg ml^-1) with or without 30 min pre-incubation with 2.5 mM H₂O₂. The data represent mean ± s.d.; n = 3 independent experiments. b, Growth curves of cells pre-cultured in SM, SM + lysine (SM + Lys, 25 μg ml^-1) or SM + methionine (SM + Met, μg ml^-1). At mid-log phase (OD_600nm ~0.5) cells were pre-incubated for 0 or 30 minutes with 2.5 mM of H₂O₂ in the respective pre-culture medium. The data represent mean ± s.d.; n = 3 biologically independent samples.

Extended data Figure 6. Effect of lysine supplementation on mammalian cell lines that are auxotrophic for lysine.

a, Growth curves showing the effect of different concentrations of lysine (0.01-10 mM) on mammalian cell growth. Percentage confluence was calculated using IncuCyte imaging system (Essen Bioscience). The data represent mean ± s.d.; n= 3 biologically independent samples. b, Intracellular lysine concentrations in different mammalian cell lines grown in DMEM with a range of concentrations of lysine (0.01-10 mM). Cells were seeded at 0.5 x 10⁶ cells per 6- cm plate and grown for 48 h before collection. The data represent mean ± s.d.; n = 3 biologically independent samples.

Extended data Figure 7. Lysine harvesting protects against H₂O₂ in different yeast and bacteria species.

a, Effect of the supplementation of lysine on the resistance of mammalian cells to H₂O₂. Cell biomass was quantified by crystal-violet staining following 24 h treatment with H₂O₂ at a range of concentrations in cultures supplemented with 0.1-10 mM lysine. The data represent mean ± s.d.; n = 3 biologically independent samples. b, Growth curves of C. tropicalis, the S. cerevisiae laboratory strain BY4741 with repaired auxotrophic loci and two non-laboratory isolates of S. cerevisiae in SM media with or without 25 μg ml^-1 lysine. When challenged with 1.5 mM H₂O₂, cultures supplemented with lysine (pre-cultures were also supplemented) can survive and eventually grow (red versus green curves). c, Spot test of the resistance of P. pastoris to H₂O₂. Overnight cultures were grown in SM media (potassium phosphate-buffered, pH 6.0) with or without lysine (76 mg l^-1) supplementation and were diluted to OD_600nm= 1 in water, serial diluted and spotted onto SM media (potassium phosphate-buffered pH 6.0) with or without lysine supplementation and H₂O₂. The experiment was repeated with similar results. d, Growth curves of B. subtilis 168 trpC2 when challenged with 1.5 mM H₂O₂ versus a water control, at mid-log phase in S7 minimal media with or without lysine (40 μg ml^-1) supplementation.

Extended data Figure 8. Yeast can accumulate high levels of cadaverine and aminopropylcadaverine that are not toxic.

a,b, Cadaverine (a) and aminopropylcadaverine (APC) (b) concentrations in S. cerevisiae wild-type strain overexpressing E. coli LdcC (WT LDC) or carrying the empty plasmid (WT pYX212). Polyamines were derivatized with dansyl chloride and quantified by LC-MS/MS. The data represent mean ± s.d.; n = 3 biologically independent samples. Unpaired two-tailed Student’s t-test: wild-type pYX212 versus WT LDC in non-supplemented conditions *** P = 0.0003 and supplemented conditions *** P = 0.0007 for a; and **P = 0.0054 and ***P = 0.0007 for non-supplemented and supplemented conditions for b. c, Growth curves showing the effect of lysine (250 μg ml^-1) on growth when wild-type strain is overexpressing E. coli LdcC. The data represent mean ± s.d.; n = 3 biologically independent samples.

Extended data Figure 9. Cadaverine can partially substitute for canonical polyamines, but only at non-physiologically high concentrations and at a certain pH, and does not protect S. cerevisiae against H₂O₂ stress.

a,b, Growth curves of Δspe1 strain depleted from spermidine grown in SM media alone or supplemented with 250 mM cadaverine in the absence (a) or presence of 0.1 mM spermidine (b). The data represent mean ± s.d.; n = 3 biologically independent samples. c, Growth curves showing the effect of lysine supplementation on wild-type and Δspe1 strains carrying a control plasmid (empty) or overexpressing E. coli LdcC (LDC), when grown in SM media buffered or not buffered at pH 5.0. The data represent mean ± s.d.; n = 4 biologically independent samples per experiment. The experiment was repeated twice. d, H₂O₂ tolerances were determined as described above, but substituting lysine with 5 mM cadaverine. The data represent mean ± s.d.; n = 4 independent experiments. Even in the presence of this high level of cadaverine, the yeast cells to not tolerate higher levels of H₂O₂.

Figure 1. A promiscuous reaction of Spe1p forms cadaverine from lysine in yeast

a. Lysine biosynthetic enzymes are differentially induced upon exposure to 1.5 mM H₂O₂ and deletion of TPO1 (Δtpo1). Shown is a 210-min times series recorded by SWATH-MS and re-processed with Spectronaut Pulsar X. Expression levels are shown as log₂ (differentially expressed enzymes). b, The decarboxylation of lysine leads to the formation of cadaverine c, Cadaverine concentrations in overnight wild-type (WT) and Δspe1-Δspe4 yeast cultures with or without lysine (25 μg ml^-1) supplementation. Mean ± s.d., n = 4 biologically independent samples except for wild-type non-supplemented and Δspe2 supplemented, for which n = 3. Unpaired two-tailed Student’s t-test versus the non-supplemented control: *P = 0.0163, **P = 0.0045, ****P ≤ 0.0001. d, Schematic illustration of the canonical yeast polyamine biosynthesis pathway. ODC, ornithine decasboxylase e, ¹³C₅ ¹⁵N₂ cadaverine is formed from isotope-labelled lysine ([¹³C₆ ¹⁵N₂] lysine) after a 1-h incubation of wild-type strains, but is not formed in Δspe1 cells. This experiment was repeated three times with similar results. f, Results of docking lysine (red) and ornithine (green) to the same site of Spe1p using Glide from Schrödinger suite (v. 2015-3) and a homology model. g, h, In vitro activity of Spe1p on metabolizing ornithine (g) and lysine (h) (mean ± s.d.; n = 3 independent experiments). The saturation curves were fitted to a Michaelis-Menten equation.

Figure 2. Lysine harvest in Saccharomyces cerevisiae.

a, Lysine concentrations in wild-type and Δtpo1 yeast grown in synthetic media supplemented with or without lysine (25 μg ml^-1). The cells were grown until the mid-log phase and analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) (mean ± s.d.; n = 4 biologically independent samples except for Δtpo1, for which n = 3). Unpaired two-tailed Student’s t-test versus the non-supplemented control: **P = 0.0056 for wild-type and P = 0.0022 for Δtpo1. b, Harvesting to reach extreme concentrations is specific for lysine. Amino acid concentrations in wild-type yeast grown in synthetical minimal (SM) media supplemented with individual amino acids (left) or rich media (yeast extract; right) plotted as fold change compared to the average profile of seven replicates in unsupplemented synthetic media (mean ± s.d., n = 4 biologically independent samples). c, Quantification of extracellular lysine during the growth of prototrophic (LYS2) and auxotrophic (Δlys2) yeast in SM media supplemented with lysine (SM+Lys) or synthetic complete media (SC). Cultures were initiated with an OD_600nm of 0.125 and grown for 30 h. Samples were taken every 3 h, collected and the supernatant was used for quantification. Mean ± s.d.; n = 4 biologically independent samples.

Figure 3. Lysine harvesting increases the tolerance of yeast to oxidants by replenishing NADPH and increasing glutathione levels.

a, Lysine harvesting replenishes the pools of NADPH and reduced glutathione (GSH). The scheme shows the flux balance analysis upon maximizing the glutathione oxidoreductase reaction b, Lysine harvesting complements the methionine auxotrophy of Δzwf1 cells. Cells were grown with or without lysine (100 μg ml^-1) or methionine (40 μg ml^-1) (mean ± s.d.; n = 4 biologically independent samples). c, d, The availability of NADPH increases in lysine harvesters. The redox state of NADPH/NADP⁺ was assessed by a fluorescence biosensor assay, using the fluorescent proteins GFP and mKate2, in plasmid-complemented prototrophs (c) and auxotrophs (d) supplemented with histidine, leucine and methionine. Mean ± s.d.; n = 4 independent experiments. e, GSH concentrations, measured as a conjugate with N-ethylmaleimide (GSH-NEM) to prevent oxidation, increase in lysine harvesters. Mean ± s.d. (n = 3 biologically independent samples for wild type and n = 4 for lysine-supplemented wild-type). Unpaired two-tailed Student’s t-test *P = 0.0426. f, Spot test assessing the diamide resistance (0-1.5 mM) of wild-type and Δzwf1 cells supplemented with methionine (40 μg ml^-1) or lysine (50 μg ml^-1) (n=3) g, L-Lysine harvesting increases tolerance to H₂O₂, but exposure to D-lysine has adverse effects. Cells were pre-cultured in the presence of L-lysine and D-lysine and exposed to H₂O₂ for 16 h. The tolerance to H₂O₂ stress was assayed by density (OD_600nm). Mean ± s.d.; n = 4 independent experiments. h, Quantification of reactive oxygen species (as assessed by dihydrorhodamine staining) in wild-type cells and lysine harvesters (50 μg ml^-1) incubated with 10 mM H₂O₂ for 1 h (mean ± s.d.; n = 3 independent experiments). Unpaired two-tailed Student’s t-test *P = 0.0123.