Investigating acetate overflow in Saccharomyces cerevisiae

This repository contains the COPASI models and experimental concentration time courses used to quantify acetate and ethanol fluxes in Saccharomyces cerevisiae in the manuscript:

Bidirectional acetate exchange couples ethanol turnover to acetyl-CoA metabolism in Saccharomyces cerevisiae
Anthony Carneiro, Mohammad Zishaan Kodooruth, Hanna Kulyk, Gilles Truan, Hélène Martin-Yken, Pierre Millard and Sara Castaño-Cerezo.

The study combines isotope tracing and metabolic flux analysis to resolve simultaneous acetate production and assimilation during sugar utilization. It examines how acetate exchange responds to changes in hexose uptake, fermentative and respiratory metabolism, deletion of ALD6, and expression of an acetate-to-mevalonate pathway. Tracing extracellular ethanol also reveals its contribution to acetate production during net ethanol overflow.

Repository contents

The repository contains 51 COPASI files and 51 experimental data tables, covering 17 combinations of experimental condition and growth phase, each with three biological replicates.

File or directory	Contents
Figure 1/	Wild-type cultures on glucose with 5 mM [U-¹³C]acetate.
Figure 2/	Glucose-uptake perturbations with 2-deoxyglucose or MTH1-ΔT, and galactose/acetate cultures of wild-type, Δald6 and pathway-expressing strains.
Figure 3/	[U-¹³C]ethanol-tracing experiments in wild-type and Δald6 strains, with separate fermentative and respiratory phases.
description.xlsx	Experiment index linking file identifiers to biological replicates, medium composition, strain, growth phase and manuscript figures.
*.cps	COPASI files containing model equations, parameter values, fitting bounds, experimental-data mappings, weights and analysis settings.
*.txt	Tab-delimited concentration time courses used for parameter estimation.
LICENSE	GNU Affero General Public License, version 3.

The directories group analyses by manuscript figure. Some datasets also support other main or supplementary figures. The tables below provide an overview, and description.xlsx gives the detailed figure-to-experiment mapping.

These files focus on metabolic flux estimation.

Experimental conditions and file guide

All strains derive from S. cerevisiae CEN.PK2-1D. WT denotes wild type, Δald6 denotes deletion of ALD6, MTH1-ΔT denotes the strain expressing the internally truncated MTH1 variant, and 2DG denotes 2-deoxyglucose. The folder label KO ALD6 + p114 identifies the Δald6 strain carrying pI2M114, which encodes the synthetic acetate-to-mevalonate pathway.

In the tables, <r> is the biological replicate number (1, 2 or 3). File identifiers are shown for the .txt data tables. The corresponding .cps file is in the same directory.

Medium concentrations below describe the nominal culture supplementation. Each data table contains the measured concentrations over the fitted growth interval. All concentrations listed in the condition tables are in mM.

Figure 1: acetate exchange during growth on glucose

Directory	Data filename	Strain	Medium
Figure 1/	g6.A.<r>.txt	WT	55 glucose + 5 [U-¹³C]acetate

These experiments support Figure 1C–E and are also used in the flux comparisons in Figure 2 and Supplementary Figures 2–3.

Figure 2: inhibition of glucose phosphorylation

Directories are relative to Figure 2/Figure 2. 2DG/. All cultures contain 55 mM glucose and 60 mM [U-¹³C]acetate.

Directory	Data filename	Strain	Added 2DG (mM)
0mM 2DG/	g6.B.<r>.txt	WT	0
5mM 2DG/	g6.C.<r>.txt	WT	5
10mM 2DG/	g7.A.<r>.txt	WT	10
5mM ALD6 2DG/	g7.B.<r>.txt	Δald6	5

The WT series supports Figure 2A–C; the Δald6 series is included in the supplementary flux comparisons.

Figure 2: galactose/acetate cultures

Directories are relative to Figure 2/Figure 2. Galactose acetate/. All cultures contain 55 mM galactose and 60 mM [U-¹³C]acetate.

Directory	Data filename	Strain	Growth phase
Fermentative/WildType/	g7.C.<r>.txt	WT	Fermentative
Respiratory/WildType/	g7.Crespi.<r>.txt	WT	Respiratory
Fermentative/KO ALD6/	g7.D.<r>.txt	Δald6	Fermentative
Respiratory/KO ALD6/	g7.Drespi.<r>.txt	Δald6	Respiratory
Fermentative/KO ALD6 + p114/	g7.E.<r>.txt	Δald6 + pI2M114	Fermentative
Respiratory/KO ALD6 + p114/	g7.Erespi.<r>.txt	Δald6 + pI2M114	Respiratory

The WT series supports Figure 2A–C,F. Comparisons of WT and Δald6 support Supplementary Figure 8, and comparisons of Δald6 with and without pI2M114 support Supplementary Figures 9–10 and the interpretation of the mevalonate experiments.

Figure 2: reduced glucose uptake in the MTH1-ΔT strain

Directories are relative to Figure 2/Figure 2. Mth1/. Both phases are from cultures containing 55 mM glucose and 60 mM unlabeled acetate.

Directory	Data filename	Strain	Growth phase
Phase 1/	g5.A.<r>.txt	MTH1-ΔT	First phase
Phase 2/	g5.A2.<r>.txt	MTH1-ΔT	Second phase

These experiments support the flux comparisons in Figure 2A–C and Supplementary Figures 2–3.

Figure 3: ethanol tracing

Directories are relative to Figure 3/. All cultures contain 55 mM galactose, 60 mM unlabeled acetate and 10 mM [U-¹³C]ethanol.

Directory	Data filename	Strain	Growth phase
A fermentative/	g8.A.<r>.txt	WT	Fermentative
A respi/	g8.Arespi.<r>.txt	WT	Respiratory
B fermentative/	g8.B.<r>.txt	Δald6	Fermentative
B respi/	g8.Brespi.<r>.txt	Δald6	Respiratory

The fermentative WT and Δald6 datasets support Figure 3A and 3B, respectively, and Supplementary Figure 7. Both growth phases are included in the supplementary flux comparisons.

Data-table format

All .txt files have the same nine columns, separated by tabs, with a header in the first row.

Column	Meaning	Unit
experiments	Experiment identifier used in the data table; the filename identifies the biological replicate.	—
time	Elapsed time within the fitted growth interval.	h
X	Biomass concentration.	gDCW L⁻¹
Glc	Extracellular hexose concentration: glucose or galactose, according to the condition.	mM
EthC12	Unlabeled extracellular ethanol.	mM
AcC12	Unlabeled extracellular acetate.	mM
AcC13	¹³C-labeled extracellular acetate.	mM
EthC13	¹³C-labeled extracellular ethanol.	mM
Gly	Extracellular glycerol.	mM

gDCW means grams of dry cell weight. Biomass was calculated from optical density as X = OD600 / 3.416, as described in the manuscript.

Model and flux definitions

The models describe central carbon metabolism and extracellular concentration dynamics, with unlabeled and labeled pools for isotope tracing. They distinguish acetate production through aldehyde dehydrogenase (Ald) from assimilation through acetyl-CoA synthetase (Acs), and represent forward and reverse alcohol dehydrogenase (Adh) fluxes separately. Ethanol evaporation is also represented.

Biomass-specific metabolic fluxes are assumed constant within each fitted growth phase, while biomass and extracellular concentrations vary over time. Each file defines 14 fitted parameters, including fluxes, growth rate and initial concentrations. Parameter estimation minimizes a weighted sum of squared residuals, with he column mappings, weights, parameter bounds and starting values stored in each file.

Key COPASI global quantities are:

Quantity	Interpretation
growth_rate	Specific growth rate, in h⁻¹.
v_glc_uptake	Hexose uptake flux.
v_ald_f	Acetate formation through Ald.
v_acs_f	Acetate assimilation through Acs.
v_adh_f	Ethanol formation from acetaldehyde through Adh.
v_adh_r	Ethanol oxidation to acetaldehyde through Adh.
v_pdc_f	Pyruvate decarboxylase flux.
v_Ace_net	Net acetate flux: v_ald_f - v_acs_f.
v_EtOH_net	Net metabolic ethanol flux: v_adh_f - v_adh_r; evaporation is represented separately.

Metabolic fluxes are expressed in mmol gDCW⁻¹ h⁻¹. Positive net exchange denotes production and negative net exchange denotes consumption. Opposing fluxes can therefore remain substantial even when net exchange is close to zero.

The experimental organism and condition are identified by this README, the directory and description.xlsx.

Using the models

Software

The files were saved with COPASI 4.39 (build 272), the version used for the analyses described in the manuscript. COPASI installers and documentation are available from the official COPASI website. A spreadsheet viewer can be used to read description.xlsx; the data tables can be opened with a text editor or imported into other analysis software.

Example: fit one biological replicate

1. Download the repository while preserving the directory structure, or clone it:

   git clone https://github.com/MetaSys-LISBP/Yeast_acetate.git
   cd Yeast_acetate

2. Open Figure 1/g6.A.1.cps in COPASI.

3. Open Parameter Estimation, then Experimental Data. Check that the experiment uses g6.A.1.txt from the same directory. If necessary, select this file manually. Retain the saved tab separator, header row, column mappings and weights.

4. Review the fitted parameters, bounds and Particle Swarm settings. The saved files specify an iteration limit of 4,000, a swarm size of 50, randomized starting values and a random-number seed of 0.

5. Run Parameter Estimation and inspect the Result view to compare fitted concentration profiles with the experimental data and review the estimated parameters and fit statistics.

6. To save numerical results, assign an output filename through the task's Report settings. Save any modified file under a new name to retain the supplied settings.

7. Repeat with replicates 2 and 3 and the other conditions of interest. Each file fits one biological replicate independently.

Particle swarm optimization is stochastic, and a seed of 0 requests an automatically selected seed. Repeated fits may therefore return different parameter values. The saved files retain the analysis configuration; numerical agreement should be assessed together with fit quality.

See the guides to experimental-data configuration, parameter-estimation results and particle swarm options for further details.

Citation and contact

Please cite the associated manuscript listed above when using these models or data. The manuscript provides the experimental procedures, biological interpretation and supplementary analyses.

For questions, contact the corresponding authors:

• Pierre Millard: millard@insa-toulouse.fr
• Sara Castaño-Cerezo: castanoc@insa-toulouse.fr

License

This repository is distributed under the GNU Affero General Public License, version 3. See LICENSE for the full terms.