# ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks

Pierre Millard, Uwe Schmitt, Patrick Kiefer, Julia Vorholt, Stéphanie Heux, Jean-Charles Portais

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Pierre Millard, Uwe Schmitt, Patrick Kiefer, Julia Vorholt, Stéphanie Heux, et al.. ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks. PLoS Computational Biology, 2020, 16 (4), pp.e1007799. 10.1371/journal.pcbi.1007799. hal-04976157

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1 ScalaFlux: a scalable approach to quantify fluxes in metabolic subnetworks

2

3 Pierre Millard¹, Uwe Schmitt², Patrick Kiefer³, Julia A. Vorholt³, Stéphanie Heux¹ and Jean-

4

Charles Portais¹,⁴,⁵,*

5

6 ¹TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France

7 ²Scientific IT Services, ETH Zurich, 8092, Zurich, Switzerland

8 ³Institute of Microbiology, Department of Biology, ETH Zurich, 8093, Zurich, Switzerland

9 ⁴MetaToul-MetaboHUB, National infrastructure of metabolomics and fluxomics, Toulouse,

10 France

11 ⁵STROMALab, Université de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, Toulouse,

12 France

13

14 *Corresponding author

15 E-mail: jean-charles.portais@insa-toulouse.fr

16

17 Short title: ScalaFlux: scalable isotope-based metabolic flux analysis
18 Abstract

19

20 ¹³C-metabolic flux analysis (¹³C-MFA) allows metabolic fluxes to be quantified in living
21 organisms and is a major tool in biotechnology and systems biology. Current ¹³C-MFA
22 approaches model label propagation starting from the extracellular ¹³C-labeled nutrient(s),
23 which limits their applicability to the analysis of pathways close to this metabolic entry point.

24 Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of
25 interest by modeling label propagation directly from the metabolic precursor(s) of this
26 subnetwork. The flux calculations are thus purely based on information from within the
27 subnetwork of interest, and no additional knowledge about the surrounding network (such as
28 atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required.

29 This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up
30 from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits
31 compared with current ¹³C-MFA approaches: greater network coverage, lower data
32 requirements, independence from cell physiology, robustness to gaps in data and network
33 information, better computational efficiency, applicability to rich media, and enhanced flux
34 identifiability. We validated ScalaFlux using a theoretical network and simulated data. We
35 also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of
36 *Saccharomyces cerevisiae* mutants engineered to produce phytoene, using a dataset for which
37 fluxes could not be calculated using existing approaches. A broad range of metabolic systems
38 can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the
39 analysis of metabolic fluxes.
40 Author Summary

41

42 Metabolism is a fundamental biochemical process that enables all organisms to operate and
43 grow by converting nutrients into energy and ‘building blocks’. Metabolic flux analysis
44 allows the quantification of metabolic fluxes *in vivo*, i.e. the actual rates of biochemical
45 conversions in biological systems, and is increasingly used to probe metabolic activity in
46 biology, biotechnology and medicine. Isotope labeling experiments coupled with
47 mathematical models of large metabolic networks are the most commonly used approaches to
48 quantify fluxes within cells. However, many biological questions only require flux
49 information from a subset of reactions, not the full network. Here, we propose a new approach
50 with three main advantages over existing methods: better scalability (fluxes can be measured
51 through a single reaction, a metabolic pathway or a set of pathways of interest), better
52 robustness to missing data and information gaps, and lower requirements in terms of
53 measurements and computational resources. We validate our method both theoretically and
54 experimentally. ScalaFlux can be used for high-throughput flux measurements in virtually any
55 metabolic system and paves the way to the analysis of dynamic fluxome rearrangements.
## 56 Introduction

Metabolic flux analysis (MFA) with stable isotope tracers, typically a ¹³C-labeled carbon source, allows intracellular fluxes to be quantified in a wide range of organisms and is now a major tool in the fields of biotechnology [1-3], systems biology [4-6] and medicine [7, 8].

Current approaches rely on isotopic models to simulate tracer propagation through metabolic networks [1, 9-14]. Fluxes are then estimated by fitting experimental concentrations and isotopic profiles of metabolites. Current simulation frameworks require known and constant label input(s). The only constant label input(s) is (are) the isotopically-labeled nutrient(s) in the extracellular medium, which must therefore be included in the flux model. This requirement also applies to alternative ¹³C-MFA frameworks, such as metabolic flux ratio analysis [15, 16] and kinetic flux profiling [17]. In practice, this means that all metabolic models must explicitly include the labeled nutrient(s) initially provided to the biological system and all the pathways that distribute the isotopic tracer up to the pathway of interest. To ensure fluxes are identifiable, the extracellular fluxes and the intracellular concentrations and labeling of upstream metabolites must also be measured. This is a major limitation for investigating i) pathways far downstream of the labeled nutrient(s), ii) networks with reaction gaps (e.g. an uncertain network topology), iii) incomplete datasets, iv) experiments performed in rich media, or v) situations where the isotopic transitions remain uncertain or complex (e.g. ²H tracer) [1, 18]. This also makes the entire experimental and computational workflow very time consuming, costly and error prone. Overall, the modeling requirement that the tracer has to be propagated right from the extracellular nutrient limits the application of flux measurements to pathways closely related to the label input. The vast majority of existing ¹³C-flux studies focus indeed on central carbon metabolism, and most ¹⁵N-flux studies focus on
80 the nitrogen assimilation network [1, 4, 6, 17, 19]. There is therefore a need for more robust
81 and scalable approaches to quantify metabolic fluxes in biochemical systems.

82 Here, we propose a new isotope-based-MFA approach, named ScalaFlux, to measure fluxes at
83 the level of any metabolic subnetwork of interest, in which label propagation is modeled
84 directly from the metabolic precursor(s) of this subnetwork. ScalaFlux uses a limited amount
85 of input data and increases the number of pathways that can be accessed, while significantly
86 reducing experimental and computational requirements. We demonstrate the value of
87 ScalaFlux with *in silico* simulations and its practical applicability by quantifying *in vivo*
88 fluxes in the yeast prenyl pyrophosphate pathway.
89

## 90 Results

91
92 *Basic principle: reconsidering label inputs*

93 Understanding the basic principle of the proposed approach requires some concepts and
94 terminology that are introduced and illustrated using the example network shown in Fig 1.
95 This network of 18 metabolites and 20 reactions includes three topological motifs classically
96 found in metabolism: a linear pathway, a branching node and a cycle. We refer to the initial
97 source(s) of label – i.e. the extracellular nutrient(s), here Xout – as the *global label input(s)* for
98 the metabolic network. After Xout is switched from natural abundance to isotopically labeled,
99 the isotopic tracer propagates through the metabolic network and the intracellular metabolites
100 (Xin, A, ..., O), which are progressively labeled as a function of metabolite concentrations and
101 fluxes. Fluxes can then be estimated using a model-based approach by minimizing the
102 difference between experimental labeling data and the labeling profiles simulated by the
103 model.
104

104
105

<figure><img src="image_2.png" /><figcaption>**Fig 1. Principle of ScalaFlux.** Panel A shows an example network in the Systems Biology Graphical Notation format (SBGN, www.sbgn.org) [20] to illustrate the basic principle of ScalaFlux. The flux models (and associated datasets) required to quantify the flux through reaction r16 using classical non-stationary ¹³C-MFA and ScalaFlux are compared in panel B. The ScalaFlux model, the set of measurements required for the flux calculation, and the flux calculation workflow are shown in panel C.</figcaption></figure>

112

Current non-stationary ¹³C-flux calculation frameworks require constant label input(s) so the global label input(s) must be included in the flux models. To specifically measure the flux through reaction r16 in the example network, the flux model (red boundaries in Fig 1A) must contain $X_{out}$ and all the reactions that contribute to isotope propagation up to the product of r16. This flux model includes a total of 17 reactions, 1 (global) label input and 14 metabolic intermediates (Fig 1B). Measurements of metabolite concentrations and labeling at several nodes of the network as well as of extracellular fluxes and biomass composition are required to calculate the flux.
121 We propose a more scalable ¹³C-flux approach, named ScalaFlux for SCALAble metabolic
122 FLUX analysis, to quantify fluxes through a subnetwork of interest using internal information
123 from this network only. ScalaFlux does not require data on the extracellular labeled nutrient,
124 upstream metabolites, or any other knowledge about the surrounding network. The flux model
125 encodes a metabolic subsystem (i.e. a subset of the cellular metabolic network) and
126 specifically contains the reaction(s) of interest, as illustrated in Fig 1 and described in detail
127 below. All the metabolic substrates in this subsystem are considered local label inputs, and
128 label propagation is simulated directly from these local label inputs. If the reaction of interest
129 is r16, the labeling dynamics of *M* is defined as the local label input of the corresponding
130 subsystem to simulate the labeling dynamics of *N*. In contrast to global label inputs, which are
131 constant, known and controlled, the labeling of local label inputs changes with time, is not
132 known *a priori* and cannot be controlled. Label incorporation can nevertheless be determined
133 experimentally and be used for the downstream reactions. Using these discrete measurements
134 as direct label inputs for simulations would result in sharp changes in label input at each
135 measurement time and thereby yield stiff equations and simulation artifacts. The first step of
136 the ScalaFlux workflow (Fig 1C) therefore consists in transforming the discrete measurements
137 into a continuous (time-dependent) representation by fitting analytical functions, ensuring
138 smooth variations as a function of time. A system of ordinary differential equations (ODEs)
139 can then be constructed using conventional frameworks to simulate label propagation from
140 the local label input(s). By combining this simulation approach with optimization routines,
141 fluxes can be estimated by fitting experimental data. This workflow has been implemented in
142 a major update of IsoSim [21] (see Methods for details).

143 Importantly, the studied subsystem can include larger parts of the network, as detailed in the
144 following sections. This means that any given (set of) flux(es) can be quantified
145 independently of the rest of the metabolic network, with no additional measurements
146 (extracellular fluxes, growth rates, biomass composition, concentrations and labeling of
147 upstream metabolites), and independently of the (often incomplete) knowledge of the
148 metabolic network outside the boundaries of the subsystem under study.

149 ScalaFlux exploits many concepts from non-stationary ¹³C-MFA and thus benefits directly
150 from recent advances in the field, such as efficient mathematical frameworks for experimental
151 design [13, 14, 22-24], simulation [14, 25-27], optimization [10, 28] and sensitivity analysis
152 [14, 29]. Because it is based on detailed modeling of isotope propagation, ScalaFlux is generic
153 with respect to the network topology (flux models can include branching nodes, cycles, or any
154 other of the topological motifs that compose metabolic networks), the isotopic tracer (²H, ¹³C,
155 ¹⁵N, etc), and the type of isotopic measurement (MS, MS/MS, NMR, etc). The flux analyses
156 presented in the rest of the article are based on mean molecular enrichment data collected by
157 mass spectrometry in ¹³C-labeling experiments.

158

159 *Construction of flux models*

160 Flux models must precisely describe the topology of the subnetwork of interest while ensuring
161 independence from the surrounding network. A generic procedure is presented in this section
162 to streamline the construction of self-consistent flux models of any part of a metabolic
163 network.

164 We define a *minimal subsystem* S<sub>Y</sub> as the minimal set of reactions required to simulate the
165 labeling dynamics of a given metabolite *Y*. A metabolic network containing *n* metabolic
166 intermediates can thus be decomposed into *n* minimal subsystems. The minimal subsystem S<sub>Y</sub>
167 must include all the reactions that produce *Y* (since they may all affect its labeling dynamics),
168 with their substrates corresponding to local label inputs. For practical modeling reasons, a
169 sink reaction consuming *Y* has to be included to avoid its accumulation, in keeping with the
170 metabolic steady-state assumption (i.e. metabolite concentrations are constant). Each minimal
subsystem is self-consistent and can be incorporated into a flux model to estimate fluxes through the included reactions. This modular representation is the essence of the scalability of ScalaFlux. We used this procedure to decompose the example network shown in Fig 1A into 17 minimal subsystems, as shown in Fig 2A. Note that reaction r6, which is reversible, is present in two subsystems ($S_B$ and $S_C$) to account for its forward and reverse fluxes [21].

176

<figure><img src="image_3.png" /></figure>

177

**Fig 2. Network decomposition to construct flux models.** The metabolic network shown in Fig 1A can be decomposed into 17 minimal subsystems (panel A) which are sufficient to simulate the labeling dynamics of metabolic intermediates (green circles) from the local label input(s) (red circles). Each minimal subsystem is self-consistent and can be used for independent flux calculations. These minimal subsystems can also be combined to analyze larger subsystems, as shown in panels B and C.

184

To analyze larger subnetworks that include several reactions of interests, the individual minimal subsystems that compose this subnetwork should be combined (Fig 2). Two subsystems can be combined when they share a common metabolite, e.g. the two minimal subsystems $S_Y$ and $S_Z$ can be merged if $Y$ is a local label input of $Z$. The local label inputs of the resulting subsystem $S_{YZ}$ are all the local label inputs except $Y$, which is now an intermediate of $S_{YZ}$. This ensures that all the reactions (and local label inputs) that contribute
191 to the labeling dynamics of Y and Z are included. For instance, to quantify fluxes through the
192 set of reactions {r4, r9, r16, r17, r18} in the example network, S<sub>O</sub> can first be united with S<sub>N</sub>
193 (since the metabolic intermediate N is a local label input of S<sub>O</sub>) (Fig 2B), and the resulting
194 subsystem S<sub>NO</sub> can then be merged with S<sub>F</sub> (Fig 2C). The final subsystem S<sub>FNO</sub> contains all the
195 reactions of interest and has three local label inputs (A, E and M) and three intermediates (F,
196 N and O).

197

198 *Flux calculation in minimal metabolic subsystems*

199 The minimal set of measurements required to estimate fluxes in a minimal subsystem S<sub>Y</sub>
200 consists of i) the labeling dynamics of its local label input(s) (used to simulate tracer
201 propagation) and ii) the labeling dynamics of Y (used for flux estimation). These transient
202 label dynamics are thus sufficient to estimate the turnover rate of Y, i.e. the ratio between its
203 pool and its biosynthetic flux. In a branched pathway, this information is also sufficient to
204 determine the contribution of each converging reaction to the biosynthesis of Y. Absolute
205 fluxes can be estimated when the absolute concentration of Y is available. The absolute *in vivo*
206 flux through a given reaction in any linear pathway can thus be estimated from reactant data
207 alone.

208 ScalaFlux was tested on the metabolic network shown in Fig 1A. Metabolite concentrations
209 and fluxes were initialized at the values listed in the Supporting information (S1 Table), and
210 label propagation through this network was simulated to create a theoretical dataset (S1 Fig).
211 We estimated fluxes in all minimal subsystems (Fig 3A) from these theoretical labeling
212 dynamics. The transient ¹³C-enrichments of all local label inputs were accurately described by
213 fitting a double logistic function (S2 Fig), and these analytical functions were used as label
214 inputs for flux calculation.

215
<figure><img src="image_4.png" /><figcaption>Fig 3. Fluxes through each reaction of the example network (Fig 1A) estimated by analyzing all minimal subsystems. Fluxes were estimated independently in all the minimal subsystems shown in panel A. The estimated fluxes are in good agreement with the true values ($R^2 = 0.98$, p-value = $1.10^{-14}$, panel B). The distribution of fluxes estimated from 200 noisy datasets are shown in panel C, with the true value used for simulation shown as a red dot and the median of the estimated fluxes shown as a white dot.</figcaption></figure>

The labeling dynamics of metabolic intermediates are accurately fitted by the flux models for all minimal subsystems (S3 Fig). The estimated fluxes are in good agreement with the true values used to run the simulations ($R^2 = 0.98$, Fig 3B), with an average relative error of 7 %. For the reversible reaction r6, both the forward and reverse reaction rates were determined. The robustness of ScalaFlux to measurement noise was assessed by estimating fluxes from 200 datasets in which Gaussian noise was added to the theoretical data, assuming a typical precision 0.02 for ¹³C-enrichments and of 10 % for concentrations [30, 31]. The distribution of fluxes estimated from these datasets indicates that the precision of the method is good, with an average relative standard deviation of 13 % (Fig 3C). ScalaFlux is thus robust to measurement uncertainty. Overall, the proposed approach provides accurate estimates of
234 absolute fluxes, with no measurement of extracellular uptake or production fluxes having
235 been provided as input. This proof of concept example validates the proposed approach.

236

237 *From individual reactions to metabolic pathways: combining minimal subsystems enhances*
238 *flux identifiability and precision*

239 As well as quantifying fluxes in minimal subsystems, ScalaFlux can be used to analyze larger
240 subsystems. Just like in minimal subsystems, the set of measurements required to estimate
241 fluxes in larger subsystems consists of i) the labeling dynamics of local label input(s) and ii)
242 the labeling dynamics of (at least one) metabolic intermediate(s).

243 To illustrate the value of this scalability, we explored different options to estimate the flux
244 through the pathway composed of the seven reactions {r10, ..., r16} (Fig 4A). We identified a
245 total of 29 subsystems (and associated datasets, Fig 4B) that potentially enable flux evaluation
246 through this pathway. Of course, this flux can be estimated through each reaction
247 individually, as demonstrated above, corresponding to subsystems S<sub>G</sub>, S<sub>H</sub>, S<sub>IJ</sub>, S<sub>K</sub>, S<sub>L</sub>, S<sub>M</sub>, and
248 S<sub>N</sub> in Fig 4B. Several reactions in this pathway can also be combined into a single flux model
249 (following the rules defined in section *Construction of flux models*), e.g. by merging two
250 connected subsystems as done for subsystems S<sub>GH</sub>, S<sub>HK</sub>, S<sub>LM</sub> and S<sub>MN</sub>.

251
<figure><img src="image_5.png" /><figcaption>**Fig 4.** Demonstration of the scalability of ScalaFlux. The absolute flux through the pathway r10-r16 (orange reactions in panel A) can be quantified in 29 different subsystems (columns in panel B), each of which i) include different reactions (in blue) and ii) exploit different sets of measurements (labeling of local label inputs in red, and concentrations and labeling of metabolic intermediates in green). The fluxes estimated for each subsystem are shown in panel C and are compared to the true value (1, horizontal line).</figcaption></figure>
259
260 The fluxes calculated for each subsystem are shown in Fig 4C and are all close to the true
261 value (1.0). Increasing the size of the subsystem used for flux calculation improves both the
262 accuracy and precision of the estimated fluxes. For instance, the flux was estimated at
263 0.96±0.10 for the minimal subsystem S<sub>G</sub> and at 0.99±0.04 for the largest subsystem S<sub>GHIJKLM</sub>.
264 This is because the reconciliation of larger datasets during flux calculation increases the
265 robustness of the approach. Experimental and analytical efforts can thus be optimized
266 depending on the required flux precision.

267 Another advantage of this scalability is that it increases flux identifiability. For instance,
268 estimating the flux through r16 is possible via the flux model of S<sub>N</sub>, provided the labeling
269 dynamics of <i>M</i> is available (Fig 4B). If <i>M</i> cannot be measured, label propagation cannot be
270 simulated and no flux can be estimated. However, if the labeling dynamics of <i>L</i> is available,
271 the flux through r16 can still be estimated using the flux model of S<sub>MN</sub> for which the labeling
272 dynamics of the local label input <i>L</i> is known. The most appropriate flux model can thus be
273 selected based on the available data, without making the additional assumptions or
274 oversimplifications required by current approaches (e.g. using hypothetical tracer atom
275 transitions from upstream pathways, defining reversible reactions as irreversible, or lumping
276 reactions). Since each subsystem can be investigated independently of the rest of the cellular
277 network, poorly identified parts of the network (e.g. due to missing measurements or an
278 uncertain topology) do not affect the reaction(s) of interest.
279

280
*Biosynthesis of prenyl pyrophosphates in yeast*

281 ScalaFlux provides the opportunity to reconsider published datasets from which fluxes could
282 not be calculated because of the lack of an appropriate modeling framework. As an example
283 application, we analyzed a published dataset on the metabolism of prenyl pyrophosphates, the
precursors of isoprenoids, in the yeast *Saccharomyces cerevisiae* [32]. Isoprenoid biosynthesis starts with isopentenyl pyrophosphate (IPP), which is isomerized into dimethylallyl pyrophosphate (DMAPP) (Fig 5A). DMAPP is then condensed with another IPP to generate geranyl pyrophosphate (GPP). Longer prenyl pyrophosphates are built by successive condensation of IPP onto each intermediate, giving farnesyl pyrophosphate (FPP) from GPP and geranylgeranyl pyrophosphate (GGPP) from FPP.

<figure><img src="image_6.png" /><figcaption>**Fig 5.** ¹³C-metabolic flux analysis of prenyl pyrophosphate biosynthesis in *Saccharomyces cerevisiae* (wild type, S037 and S023 strains). The yeast prenyl pyrophosphate pathway contains five reactions for the successive condensation of IPP (in</figcaption></figure>

290

291

292

293
294 grey) onto each intermediate (DMAPP, FPP, GPP and GGPP) (A). The labeling dynamics of
295 IPP were fitted with a double logistic function, which was used as the local label input. Fluxes
296 were estimated by fitting the metabolite concentrations and transient ¹³C-enrichments of GPP,
297 FPP and GGPP. Experimental and fitted data are shown for each strain in panel B for the
298 labeling dynamics (dots: experimental values; lines: best fit) and in panel C for the metabolite
299 concentrations. The fluxes estimated in each strain are given with their standard deviations in
300 panel D. The GGPP demand calculated from phytoene accumulation in strain S023 is shown
301 in grey for comparison. The GGPP turnover rate estimated in each strain is shown in panel E.

302

303 The published dataset contains i) steady-state concentrations of three prenyl pyrophosphate
304 intermediates (GPP, FPP and GGPP) measured during exponential growth on glucose and ii)
305 44 transient ¹³C-enrichments following a switch from unlabeled to U-¹³C-glucose (11 time
306 points for GPP, FPP, GGPP, and combined pools of IPP and DMAPP). These data were
307 collected in three different strains designed to enhance phytoene production. The GGPP pool
308 of the wild-type (WT) metabolic chassis was first increased by constructing the strain S037,
309 which overexpresses GPP and FPP synthase (ERG20) and GGPP synthase (CrtE). A
310 heterologous phytoene synthase (CrtB from *Pantoea ananatis*) was then expressed to convert
311 GGPP into phytoene in strain S023. The pools of all intermediates were higher in strains S037
312 and S023 compared to wild type, suggesting higher fluxes, but this could not be verified
313 because fluxes could not be inferred solely from these data. We therefore used ScalaFlux to
314 estimate the *in vivo* flux through the prenyl pyrophosphate pathway in the three strains.

315 The flux model is centered on the specific pathway of interest and thus only includes the five
316 reactions shown in Fig 5A. We used a double logistic function to fit the transient labeling
317 dynamics of IPP (i.e. mean molecular ¹³C-enrichment), from which accurate analytical
318 representations were obtained (Fig 5B). This function was used as label input to estimate
319 fluxes by fitting the concentrations and dynamic ¹³C-enrichments of three other intermediates
320 (GPP, FPP and GGPP). The good agreement between simulations and measurements (Figs 5B
321 and 5C, R² > 0.98) indicates that the concentrations and isotopic data are consistent with the
322 topology defined in the model. In wild-type *Saccharomyces cerevisiae*, the GGPP
323 biosynthetic flux was estimated at 0.15±0.01 nmol/gDCW/min during exponential growth on
324 glucose (Fig 5D). It increased to 0.94±0.04 nmol/gDCW/min in strain S037, hence confirming
325 the relevance of the strain design strategy in improving the availability of GGPP, the
326 precursor of phytoene biosynthesis. The flux was similar in the phytoene producing strain
327 S023 (0.93±0.04 nmol/gDCW/min). This indicates that the increased demand for GGPP does
328 not propagate upstream and does not affect its production, in agreement with the low
329 reversibility of the prenyl transferase reactions. Importantly, we verified that the flux
330 estimated by ScalaFlux in S023 was consistent with the GGPP demand for phytoene synthesis
331 estimated from phytoene accumulation (1.33±0.16 nmol/gDCW/min, Fig 5D). The good
332 agreement between these two independent methods demonstrates that ScalaFlux provides
333 accurate flux measurements from datasets collected on just a few metabolic intermediates.
334 Finally, while qualitative interpretations suggested that the turnover rate of GGPP was stable
335 in the different strains [32], this could not be verified because the fluxes could not be
336 estimated. We therefore evaluated this hypothesis by calculating the GGPP turnover from the
337 estimated fluxes and metabolite concentrations. Results indicate that GGPP turnover (Fig 5E)
338 is indeed very similar in the three strains (WT: 12.7±0.1, S037: 14.0±1.3, S023: 11.9±0.5 min⁻¹)
339 and thus confirm quantitatively that the GGPP pool increases roughly proportionally to its
340 biosynthetic flux.

341

342 Discussion

343
344 In current ¹³C-MFA approaches, label propagation has to be modeled starting from the
345 extracellular nutrient(s), which limits their applicability to flux analysis of pathways close to
346 this nutrient. Here, we present a novel MFA framework to investigate any reaction or set of
347 reactions in a subnetwork of interest based on just a few targeted measurements in this
348 subnetwork.

349 The scalability of ScalaFlux stems from the modular decomposition of metabolic networks
350 into minimal subsystems, which can be analyzed independently or merged together to analyze
351 larger subnetworks, as demonstrated using a theoretical network and simulated data. The
352 guidelines provided to decompose a network into minimal subsystems enable intuitive
353 reasoning and facilitate experimental design (e.g. in terms of the measurements to perform),
354 which can be supported further by *in silico* simulations. It is important to note that flux
355 identifiability depends on the experimental setup used (e.g. type of isotopic data, accessible
356 measurements, sampling frequency) and on biological constraints (e.g. network topology,
357 fluxes). We refer to previous work [12-14, 22, 24, 33] for extensive discussion on these
358 topics.

359 We validated the practical applicability of ScalaFlux by reanalyzing a published dataset on the
360 metabolism of prenyl pyrophosphates, from which fluxes could not be calculated using
361 current MFA approaches. Indeed, GGPP is continuously used by different processes (such as
362 protein geranylgeranylation and membrane biosynthesis) and does not accumulate in cells. Its
363 biosynthetic flux cannot therefore be measured *in vivo* without using isotopic tracers.
364 Moreover, measuring this flux using stationary ¹³C-MFA approaches would have been
365 impossible because of the topology of the prenyl pyrophosphate pathway. Non-stationary ¹³C-
366 MFA approaches could have been used, but at much higher analytical and computational
367 costs. The underlying model would have had to include many additional reactions involved in
368 ¹³C label propagation from glucose up to IPP, i.e. at least some of the central metabolic
pathways that contribute to the labeling of acetylCoA (glycolysis, the pentose phosphate pathway, and possibly anaplerotic reactions and the TCA cycle), and the entire mevalonate pathway that produces IPP from acetylCoA. This model would thus have contained several dozen reactions, for which the associated fluxes would have had to be estimated. Our approach significantly reduces the size of the model and the number of free parameters, and thereby the computational cost of the flux calculation. Moreover, the absolute pathway flux was estimated using the metabolite concentrations and ¹³C-enrichments collected for just a few metabolites using a single LC-HRMS platform [32]. Using traditional approaches, the full model would have been undetermined - and no flux could have been estimated - without additional experimental data on key points in the upstream pathways (e.g. the glucose uptake flux, and the pools and transient ¹³C-enrichments of upper intermediates), collected with different sampling times, and analyzed with different analytical platforms. Our approach thus also reduces experimental costs and processing efforts.

ScalaFlux is fundamentally scalable, providing several different ways to quantify a given (set of) flux(es). The most appropriate flux model should be selected based on the biological question to be addressed (e.g. in terms of the fluxes to be measured or the required flux precision) and practical constraints (e.g. network knowledge or available data). For instance, fluxes through individual reactions in a linear pathway can be estimated independently using different datasets. ScalaFlux can thus potentially verify (or disprove) assumptions that are usually made in ¹³C-MFA (e.g. that all the reactions in a given linear pathway carry the same flux) and to identify gaps in the current knowledge (e.g. that an intermediate of an apparently linear pathway is actually consumed by another unknown reaction, or that the assumed network topology is not sufficient to explain the labeling dynamics of some of the intermediates).
393 ScalaFlux is also highly versatile in terms of the pathways that can be monitored. It can be
394 used to measure fluxes through virtually any metabolic subsystem of interest: a single
395 reaction, a pathway, or larger networks. Because it exploits concepts from non-stationary ¹³C-
396 MFA, ScalaFlux can be used to investigate C₁-metabolism (e.g. CO₂ fixation, methylotrophy,
397 folate metabolism). It also allows the quantification of metabolic fluxes that are currently
398 difficult to measure, e.g. in secondary metabolism (such as prenyl pyrophosphate
399 biosynthesis, as demonstrated here), or the biosynthesis of co-factors (e.g. ATP or NADPH)
400 or other global regulators (e.g. ppGpp). Its scalability offers new possibilities for high-
401 throughput flux profiling of a broad range of metabolic (sub)systems, at minimal cost and
402 effort. ScalaFlux can easily be adapted to measure fluxes through other biological processes,
403 such as protein turnover.

404 Overall, in addition to broadening the range of metabolic systems that can be investigated,
405 ScalaFlux enhances the following aspects of ¹³C-MFA: minimal data and analytical
406 requirements (fluxes can be estimated robustly from just a few measurements from the
407 metabolic subsystem of interest, which can typically be collected using a single platform since
408 closely related metabolites often have similar physico-chemical properties); independence
409 from physiology (no need to measure nutrient uptake fluxes, growth rates, or biomass
410 compositions); computational efficiency and stability (smaller equation systems with fewer
411 free parameters); short labeling times (no tracer incorporation required to reach steady state),
412 which allows dynamically changing fluxes to be probed; applicability to rich media (where
413 measuring the many extracellular fluxes and labeling patterns of all the nutrients is difficult
414 and may create computational bottlenecks); and better flux identifiability (because of its
415 intrinsic scalability and robustness to missing measurements and network gaps).

416 ScalaFlux can be applied alone or in combination with other methods to address a broad range
417 of biological questions. Combined with untargeted MS(/MS) approaches [34-36], ScalaFlux
418 paves the way to ¹³C-flux studies at the cellular level. The network coverage of untargeted
419 MS(/MS) approaches is in general low and sparse, which results in poor flux identifiability
420 when the complete dataset is integrated into metabolic reconstructions. In ScalaFlux,
421 incomplete datasets can still be exploited to estimate fluxes through subsystems, and these
422 flux measurements can be used to constrain genome scale metabolic models. Our approach
423 should also be helpful to study poorly characterized organisms, for which simulations from
424 carbon entry up to the pathway of interest may not be possible.

425 From a computational point of view, the proposed approach shares many elements with
426 traditional approaches, and is compatible with all current simulation frameworks - EMUs,
427 cumomers, fluxomers, etc - [1, 14, 25]. The approach introduced here can be implemented in
428 existing ¹³C-flux calculation software [10, 26, 28, 37] with minimal effort. As proof of
429 concept, we have implemented it in IsoSim, a versatile modeling software designed to
430 integrate proteomics, metabolomics and isotopic data with stoichiometric, kinetic, regulatory
431 and thermodynamic constraints to enhance functional analyses of metabolic systems.
432 ScalaFlux is fully compatible with kinetic modeling, and thereby offers the possibility of
433 analyzing dynamic fluxome rearrangements.

434

435 Methods

436

437 *Implementation of the ScalaFlux workflow*

438 We implemented the ScalaFlux workflow (Fig 1C) in a major update of IsoSim, an R
439 software previously developed to couple kinetic and isotopic models of metabolism [21]. The
440 source code of IsoSim v2 is freely distributed under open-source license at
441 https://github.com/MetaSys-LISBP/IsoSim/.
442 Briefly, IsoSim includes functions to i) construct flux models, ii) design isotope labeling
443 experiments, iii) define local label inputs, iv) simulate label propagation, and v) fit
444 experimental data in order to estimate fluxes. Each of these steps is explained in detail in the
445 following sections.

446 All the scripts we used to construct the models, to perform the simulations and to generate the
447 figures are provided at https://github.com/MetaSys-LISBP/IsoSim/ to ensure reproducibility
448 and reusability.

449

450 *Construction of flux models*

451 IsoSim requires the following information to construct a flux model: i) the set of reactions of
452 interest, ii) the tracer atom transitions of each reaction, and iii) the accessible isotopic data.

453 IsoSim then automatically constructs the minimal system of ordinary differential equations
454 (ODEs) required to simulate the accessible isotopic measurements. The detailed procedures
455 and algorithms we used to construct the models can be found in the initial article on IsoSim
456 [21], which has been enhanced with the EMU framework [27] to reduce the size of the
457 equation system.

458 Note that each flux can be defined either as constant or calculated using a kinetic equation
459 which may depend on metabolite concentrations. IsoSim can thereby perform both
460 stoichiometric and kinetic modelling.

461

462 *Design of isotope labeling experiments*

463 The present framework provides i) direct identification of the minimal set of label input(s)
464 that need to be measured for a given flux model, and ii) simulations for different
465 configurations (e.g. different pools, flux distributions or local label input dynamics). These
466 two features are crucial to support experimental design and ensure flux identifiability before
467 performing the experiments [22].

468

469 *Fitting local label input(s)*

470 The labeling dynamics of all the EMUs identified as local label input(s) must be measured or
471 estimated. IsoSim implements methods to convert these discrete measurements into
472 continuous analytical functions. It is important to note that neither the analytical function nor
473 the estimated parameters have any biological meaning. The aim of this step is just to define a
474 sufficiently accurate representation of the isotopic profiles of the local label input(s).

475 Experimental ¹³C-enrichment dynamics of local label input(s) can be fitted by a logistic
476 function (Eq. 1):

$$
477 \qquad y(p,t) = \frac{p_1}{1+e^{-p_2 \cdot (t-p_3)}} \qquad (\text{Eq. 1})
$$

478 where *p* is the vector of parameters to estimate (here *p*₁, *p*₂ and *p*₃), and *t* is time. We also
479 implemented a double-logistic function (Eq. 2) to fit more complex labeling dynamics, as
480 proposed by Elmore et al. [38]:

$$
481 \qquad y(p,t) = p_4 + (p_5 - p_6 \cdot t) \cdot \left( \frac{1}{1+e^{(p_7-t)/p_8}} - \frac{1}{1+e^{(p_9-t)/p_{10}}} \right) \quad (\text{Eq. 2})
$$

482 Parameter estimation is formulated as a constrained non-linear optimization problem (Eq. 3):

minimize $f(p)$

$$
483 \qquad \text{subject to } g(p) > c \hfill (\text{Eq. 3})
$$

484 where *p* is the parameter vector, *f* is the objective function that evaluates the deviation
485 between the simulated and measured data, *g*(*p*) is the constraint function, and *c* is the
486 constraint vector. The objective function *f* (Eq. 4) is defined as the sum of squared weighted
487 errors:

$$
488 \qquad f(p) = \sum_i \left( \frac{x_i - y_i(p, t_i)}{\sigma_i} \right)^2 \qquad (\text{Eq. 4})
$$
where $x_i$ is the experimental value of data point $i$ collected at time $t_i$, with an experimental standard deviation $\sigma_i$, and $y_i(p, t_i)$ is the corresponding simulated value. Constraints are defined for all parameters to be estimated ($0 < p_1 < 1$, $-100 < p_2 < 100$, $-1000 < p_3 < 1000$ for the logistic function and $-1 < p_4 < 1$, $-1 < p_5 < 1$, $-10 < p_6 < 10$, $-1000 < p_7 < 1000$, $-100 < p_8 < 100$, $-1000 < p_9 < 1000$, $-100 < p_{10} < 100$ for the double logistic function) to improve convergence by reducing the solution space. The optimization problem is first solved using particle swarm optimization (R 3.2.4, pso package v1.0.3), followed by an L-BFGS-B [39] search (with an upper limit of 1000 iterations) to improve convergence. A plot of measured versus fitted data is generated to allow visual inspection of the quality of fit, and the analytical functions describing local label inputs are provided as output.

499

500 *Simulation of label propagation*

IsoSim solves the ODE system to simulate label propagation through the metabolic subnetwork of interest, using as input i) the constructed model, ii) the analytical functions describing local label input(s), iii) the metabolite concentrations, and iv) the fluxes. The simulation engine is based on the fluxomer framework [25], as detailed in [21], and has been enhanced using the EMU framework [27]. This facilitates the identifiability analysis while significantly reducing the size of the equation system to be solved.

507

508 $^{13}$C-flux calculation

Fluxes are estimated by fitting experimental data (the concentrations and labeling dynamics of metabolic intermediates). The objective function $h$ (Eq. 5) is defined as the sum of squared weighted errors [40]:

$$h(v, m) = \sum_i \left( \frac{x_i - y_i(v, m)}{\sigma_i} \right)^2 + \sum_j \left( \frac{n_j - m_j}{\sigma_j} \right)^2 \quad (\text{Eq. 5})$$
where $v$ is the vector of fluxes, $m$ is the vector of metabolite concentrations $m_j$, $x_i$ is the experimental value of the labeling at data point $i$, with experimental standard deviation $\sigma_i$, $y_i(v,m)$ is the corresponding simulated value, $n_j$ is the experimental concentration of metabolite $m_j$ with standard deviation $\sigma_j$. Equality and inequality constraints can be defined for the fluxes (default constraints: $-10^3 < v < 10^3$) and metabolite concentrations (default constraints: $10^{-6} < m < 10^3$). The objective function $h$ is minimized using the nlsic optimization algorithm [10] (with 50 iterations). The goodness-of-fit is evaluated using a chi-square test, and the mean, median, standard deviation and 95% confidence intervals of the calculated fluxes are estimated using Monte-Carlo sensitivity analysis.

522

## 523 Author contributions

Funding acquisition, J-C.P. and J.A.V.; Conceptualization and design, P.M., U.S., P.K., J.A.V., S.H., and J-C.P.; Investigation, P.M., U.S., P.K., and J-C.P.; Methodology, P.M., U.S., P.K., and J-C.P.; Software, P.M.; Visualization, P.M.; Writing - original draft preparation, P.M.; Writing - review & editing, P.M., U.S., P.K., J.A.V., S.H., and J-C.P.; Supervision, J-C.P. All authors read and approved the final version of this manuscript.

529

## 530 Funding

This work was supported by the French National Research Agency project ENZINVIVO (ANR-16-CE11-0022) and ETH Zurich.

533

## 534 Acknowledgements

We thank MetaToul (Metabolomics & Fluxomics Facilities, Toulouse, France, www.metatoul.fr), which is part of the MetaboHUB-ANR-11-INBS-0010 national infrastructure (www.metabohub.fr), for providing free access to its computational resources.

We thank Matthieu Guionnet for technical assistance and Baudoin Delépine for insightful
539 comments on the manuscript. JCP is grateful to the INSERM for funding his temporary full-
540 time researcher position.

541

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682 **Supporting Information**

683 **S1 Table. Initial values of fluxes and metabolite concentrations.** Values of fluxes and
684 metabolite concentrations used to simulate label propagation through the example network
685 shown in Fig 1A.

686 **S1 Fig. Simulation results.** Labeling dynamics in response to a switch from unlabeled $X_{out}$ to
687 fully labeled $X_{out}$.

688 **S2 Fig. Fit of local label inputs.** The labeling dynamics of the local label inputs in all the
689 subsystems shown in Fig 3 were fitted with analytical functions. The dots represent the fitted
690 data and the lines represent the best fits.

691 **S3 Fig. Flux calculation results.** Fluxes were estimated by fitting the labeling dynamics of
692 the metabolic intermediates of all the subsystems shown in Fig 3. The dots represent the fitted
693 data and the lines represent the best fits.