Metabolic Engineering 75 (2023) 12–18

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Metabolic Engineering
journal homepage: www.elsevier.com/locate/meteng

Design and application of a kinetic model of lipid metabolism in

Saccharomyces cerevisiae
Shekhar Mishra a, Ziyu Wang b, Michael J. Volk a, Huimin Zhao a, b, c, *
a
Department of Chemical and Biomolecular Engineering, Department of Energy Center for Advanced Bioenergy and Bioproducts Innovation, Carl R. Woese Institute for
Genomic Biology, USA
b
Department of Biochemistry, USA
c
Departments of Chemistry and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Lipid biosynthesis plays a vital role in living cells and has been increasingly engineered to overproduce various
Lipid metabolism lipid-based chemicals. However, owing to the tightly constrained and interconnected nature of lipid biosynthesis,
Kinetic model both understanding and engineering of lipid metabolism remain challenging, even with the help of mathematical
Free fatty acid
models. Here we report the development of a kinetic metabolic model of lipid metabolism in Saccharomyces
Fatty alcohol
cerevisiae that integrates fatty acid biosynthesis, glycerophospholipid metabolism, sphingolipid metabolism,
storage lipids, lumped sterol synthesis, and the synthesis and transport of relevant target-chemicals, such as fatty
acids and fatty alcohols. The model was trained on lipidomic data of a reference S. cerevisiae strain, single
knockout mutants, and lipid overproduction strains reported in literature. The model was used to design mutants
for fatty alcohol overproduction and the lipidomic analysis of the resultant mutant strains coupled with model-
guided hypothesis led to discovery of a futile cycle in the triacylglycerol biosynthesis pathway. In addition, the
model was used to explain successful and unsuccessful mutant designs in metabolic engineering literature. Thus,
this kinetic model of lipid metabolism can not only enable the discovery of new phenomenon in lipid metabolism
but also the engineering of mutant strains for overproduction of lipids.

1. Introduction enhancing our understanding of host metabolism and designing meta­

Growing concerns of petrochemical resource depletion and climate
change trends have, in recent times, increased the push towards sus­
tainable manufacturing of chemicals and fuels via biological routes
(Nielsen and Keasling, 2016). Numerous studies in recent years have
focused on increasing production of industrial chemicals from the lipid
metabolism such as free fatty acids, fatty alcohols, triglycerides and even
oleochemicals (d’Espaux et al. (2017); Runguphan and Keasling, 2014).
However, due to the highly constrained and regulated nature of the lipid
metabolic network, engineering the lipid metabolism even in
well-studied model organisms such as Saccharomyces cerevisiae has
invariably proved to be non-trivial and often challenging. Mathematical
modeling can play a significant role in bridging this gap by offering a
framework to integrate prior knowledge of metabolic topology as well as
data from new experiments into a single holistic compendium. For
example, the use of genome-scale stoichiometric models (GEMs) in
bolic engineering strategies has already been well documented (Sánchez
and Nielsen, 2015). GEMs are, however, restricted in the information
they can capture and predict. GEM simulations to predict flux space only
show relevancy in the metabolic steady state of a microorganism. They
are also unable to account for allosteric and regulatory control on
metabolic reactions.
To address these limitations, kinetic models are employed instead.
Kinetic models use ordinary differential equations (ODEs) to capture the
dynamic trajectories of all constituent species included in the model
(metabolites, proteins, mRNA, etc.). The rate of consumption or pro­
duction of any constituent entity is captured by mathematical formulae,
such as mass-action or Michaelis-Menten kinetics. Although no GEM has
specifically been utilized towards lipid-related metabolic engineering
applications, several kinetic models have been constructed to specif­
ically focus on the pathways associated with lipid metabolism. For
example, Savoglidis and coworkers developed a lipid kinetic model that

* Corresponding author. Department of Chemical and Biomolecular Engineering, Department of Energy Center for Advanced Bioenergy and Bioproducts Inno­
vation, Carl R. Woese Institute for Genomic Biology, USA.
E-mail address: zhao5@illinois.edu (H. Zhao).

https://doi.org/10.1016/j.ymben.2022.11.003
Received 17 July 2022; Received in revised form 29 October 2022; Accepted 8 November 2022
Available online 9 November 2022
1096-7176/© 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
S. Mishra et al.
was used with inverse metabolic control analysis (IMCA) to predict the
fold-change in gene expressions as a consequence of a single gene
deletion, solely from the changes in metabolite concentrations (Savo­
glidis et al., 2016). Recently, Tsouka and coworkers developed a lipid
metabolic reconstruction that integrates with a core metabolic GEM and
was systematically reduced around the lipid biosynthetic core (Tsouka
and Hatzimanikatis, 2020). However, no lipid kinetic model has been
used to predict the effects of lipid metabolic engineering on the pro­
duction of value-added compounds.
In this work, we describe the construction of a kinetic model of lipid
metabolism in S. cerevisiae that was designed and trained to rationalize
and predict viable lipid metabolic engineering strategies. The model
connects all submodules of the lipid network that are known to play a
role towards the success or failure of metabolic engineering strategies
for lipid overproduction. The use case of the model was demonstrated by
comparing in silico predictions of metabolic engineering strategies from
prior studies. The construction of mutants for fatty alcohol over­
production led to the discovery of a futile cycle in TAG biosynthesis,
which was hypothesized by model simulations and validated by 13C
labeling experiments. Thus, the model can be used to design new mu­
tants for overproduction of lipid-based biochemicals in future studies.
2. Methods and materials
2.1. Strains, plasmids, and mutant construction
The reference yeast strain used in this study was BY4741 (MATa
his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). All mutant strains were constructed
by introducing mutations into the BY4741 genome. Genetic engineering
to construct mutants was performed using the pCRCT plasmid reported
in the HI-CRISPR methodology (Bao et al., 2015). The plasmid contains
a variant of the SpCas9 gene, called iCas9 that was used to generate
double stranded breaks in the yeast genome. To direct the iCas9 protein
to the target region, the plasmid contains a 20 bp crispr-RNA (crRNA)
that was designed using the CRISPR tool provided by Benchling. The tool
uses two scoring systems for on-target (Doench et al., 2016) and
off-target analysis (Hsu et al., 2013). In appropriate mutations, a 50 bp
linear homologous recombination (HR) donor was added along with the
crRNA-carrying pCRCT plasmid. For yeast transformation, the
commonly used lithium acetate heat shock protocol was used with a 1 h
heat shock (Gietz and Schiestl, 2007). The details of mutant construction
including guide RNA sequences and list of all strains studied can be
found in the Supplementary Information.
2.2. Yeast cultivation
All yeast cultures were carried out in a similar manner. A yeast
colony was chosen from a freshly streaked agar plate and inoculated in 2
mL YPD media (1% yeast extract, 2% peptone, 2% dextrose) and grown
for 24 h in a 30 ◦ C incubator with shaking at 250 rpm. At the 24 h mark,
the optical density (OD600) of the cell culture was measured and a new
culture with initial OD600 of 0.2 was started in either 5 mL (round-
bottom tube) or 30 mL (baffled shake flask) of synthetic complete (SC)
media with no dropouts (0.17% yeast nitrogen base, 0.5% ammonium
sulphate, 2% dextrose). The cells were then sampled at a pre-defined
timepoint or multiple timepoints of this SC growth culture based on
the nature of the experiment (timeseries or steady state sampling).
2.3. Lipidomic analysis
Lipidomics analysis of both the reference and mutant yeast strains
was performed using a two-step chloroform-methanol extraction as
described elsewhere (Ejsing et al., 2009; Klose and Tarasov, 2016).
Briefly, harvested cells were washed with 150 mM ammonium bicar­
bonate (ABC). Cell lysis was performed by resuspending the cells in ABC
buffer, adding 200 μL zirconium glass beads and vortexing at 2500 rpm
13
Metabolic Engineering 75 (2023) 12–18
for 30 min in a Fisherbrand MultiTube vortexer (Thermo Fisher Scien­
tific, Waltham, MA). Cell lysate equivalent to 1 OD600 unit was trans­
ferred to a new tube and ABC buffer was added to a total volume of 200
μL. To this, 10 μL of the internal lipid standard mix was added along with
1 mL of 15:1 chloroform-methanol. The tube was vortexed at 2500 rpm
for 2 h. Phase separation was achieved by centrifugation at 4000 rpm for
10 min. The lower organic layer was transferred into a new vial and
dried in a SpeedVac (Thermo Fisher Scientific, Waltham, MA). To the
remaining chloroform-methanol mix in the tube, 1 mL of 2:1
chloroform-methanol was added and the tube vortexed at 2500 rpm for
another 2 h. After a second phase separation step, the lower organic
layer was transferred into a new vial and dried in a SpeedVac. The
non-polar dried lipids from the first extraction were resuspended in 100
μL of 4:2:1 isopropanol-methanol-chloroform with 7.5 mM ammonium
formate, while the polar dried lipids from the second extraction resus­
pended in 5:1 methanol-chloroform containing 0.2 mM methylamine.
The lipid resuspensions were then analyzed using a Q-Exactive Mass
Spectrometer (Thermo Fisher Scientific, Waltham, MA) operated in the
direct infusion mode via a Triversa Nanomate chip-based robotic infu­
sion nanospray (Advion, Ithaca, NY). Details of mass spectrometric
analysis, including Q-Exactive method parameters and Nanomate infu­
sion parameters, are provided in the Supplementary Information.
2.4. Lipidomics data analysis
Identification and quantification of lipid species and classes from the
lipidomic data generated by the mass spectrometer was performed by an
in-house Python script that employed the pymzML library (Bald et al.,
2012). Raw data files were input to the pipeline after conversion to the
open source mzML format using the tool MSConvert from the software
Proteowizard (Chambers et al., 2012). After identification of lipid peaks
in the data, absolute quantification of each lipid species was performed
by normalizing to the intensity of the lipid internal standard from its
corresponding class using a single-point calibration method. The Python
files associated with this pipeline are available in the GitHub repository.
2.5. Model construction
Model construction was performed in a systematic manner, where
the topology of the lipid network was first established by referring to
databases such as BRENDA (https://www.brenda-enzymes.org/) and
LipidMaps (https://www.lipidmaps.org/). Then, each enzymatic reac­
tion was assigned a mathematical rate expression in the form of
Generalized Michaelis-Menten kinetics, also known as convenience ki­
netics (Liebermeister and Klipp, 2006). The Systems Biology Markup
Language (SBML) format was employed to store the model parameters,
metabolites and reaction rate laws (Bornstein et al., 2008). The Python
library AMICI was used for integrating the kinetic model’s ODEs over
time (Fröhlich et al., 2021).
2.6. Parameter estimation scheme
Lipidomic data generated from perturbation mutants was used to
train the kinetic model by estimating parameters that fit in silico pre­
dictions of the model to the training dataset. The parameter estimation
process closely followed a multi-start gradient-based local optimization
method as described here (Raue et al., 2013). Briefly, parameter esti­
mation was performed by generating a pre-determined number of guess
vectors. From each guess vector, a gradient-based local search was
carried out to solve an optimization problem. The optimization problem
formulated in this case was the minimization of the negative
log-likelihood function. The gradient searches from each start are in­
dependent of each other and hence, could be performed in a parallel
manner, resulting in a faster estimation procedure. The Python libraries
of AMICI (Fröhlich et al., 2021), PEtab (Schmiester et al., 2021) and
pyPESTO (Schälte et al., 2021) were employed to formulate the
S. Mishra et al.
parameter estimation problem and perform the parallelized gradient
searches. The scripts employed in the above parameter estimation
scheme as well as the PEtab files can be found in the GitHub repository
associated with this study.
2.7. Isotope labeling experiments for futile cycle analysis
Experimental confirmation of futile cycles in the fatty alcohol mu­
tants was performed via stable isotope labeling experiments using 13C6
–glucose containing media. Briefly, the control and mutant strains were
cultured in 2 mL YPD for 24 h and transferred to 5 mL SC media at a
starting OD600 of 0.2. After 24 h of growth in SC media, the cells were
washed in water and resuspended in labeled isotope SC media con­
taining 2% 13C6 – glucose. At timepoints of 1, 2, 3, 4, 8 and 24 h, OD600
values of the cultures were measured, and 5 OD600 units of cells were
harvested. After washing and cell lysis with glass beads, lipid extraction
was performed on 2 OD600 units of cell lysate, followed by lipidomic
analysis. Steps for lipid extraction and lipidomic analysis were followed
as described above.
2.8. Code availability
The Python scripts accompanying this study can be found in the
following GitHub repository: https://github.com/HuiminZhao/Mishra-
Saccharomyces-lipid-kinetic-model.
3. Results
3.1. Model construction - network topology and rate kinetics
The construction of a kinetic model of lipid metabolism in
S. cerevisiae began with a tabulation of all the important pathways
within the lipid network that are reported to play a role in either lipid
homeostasis or lipid overproduction (Klug and Daum, 2014). The list of
pathways incorporated in the model include fatty acid synthesis and
elongation, biosynthesis of glycerophospholipids, sphingolipids, storage
lipids (diacylglycerol, triacylglycerol and ergosterol esters), ergosterol
and free fatty acid synthesis and degradation (which include activation
as well as β-oxidation) as depicted in Fig. 1. A separate reaction for the
conversion of fatty acyl-CoA to fatty alcohol via the fatty acyl-CoA
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Metabolic Engineering 75 (2023) 12–18
reductase (FAR) reaction was added. Fatty acid elongation and ergos­
terol biosynthesis were modeled as lumped reactions. This was carried
out to minimize uncertainty in parameter estimation in these pathways
as quantitative data of the intermediates within these pathways could
not be obtained from the lipidomic analysis. To model the kinetic rate
expressions for each reaction, the convenience rate law was employed
(Liebermeister and Klipp, 2006). Convenience kinetics also allows for
the addition of regulatory interactions such as activation or inhibition.
Such details were borrowed from earlier models of lipid metabolism that
included regulatory information (Alvarez-Vasquez et al., 2005, 2011).
3.2. Parameter estimation with perturbation data
The first round of parameter estimation on the kinetic model was
performed using lipidomic data obtained from the reference strain of
S. cerevisiae, BY4741, grown in synthetic complete media. 19 lipid
classes were quantified using internal standards along with free fatty
acid measurements in the extracellular environment (supernatant).
Parameter estimation on the model using the collected lipidomic data
from the reference strain suggested that this dataset alone could not
suitably be used to identify all the parameters in the reference model. To
improve estimates of parameters, mutations associated with lipid
overproduction were introduced into the BY4741 strain. Specifically,
three strains associated with increased flux towards either free fatty
acids, fatty acyl-CoA or fatty alcohol were constructed, hereby referred
to as overproduction reference strains. These three strains were created
with mutations that have been reported in literature as successful stra­
tegies towards increasing titers of the corresponding products – i) triple
knockout of faa1Δ, faa4Δ and pox1Δ for free fatty acid overproduction
(scFA-4) (Runguphan and Keasling, 2014), ii) triple overexpression of
fatty acid biosynthesis genes via knock-in of TEF1 promoter (pTEF1) as
pTEF1:ACC1, pTEF1:FAS1 and pTEF1:FAS2 for fatty acyl-CoA over­
production (scFA-5) (d’Espaux et al., 2017), and iii) the pTEF1 knock-in
strain scFA-5 carrying a chromosomally-integrated heterologous copy of
the MmFAR1 gene for fatty alcohol overproduction (scFA-7) (d’Espaux
et al., 2017). The MmFAR1 expression cassette was integrated at the
chromosomal site X-4 identified in an earlier study (Mikkelsen et al.,
2012). Fig. S6 shows a lipid metabolic map with all the targeted genes
highlighted.
Network perturbation effects on lipid profiles were probed by
Fig. 1. The lipid metabolic map captured in the ki­
netic model and the essential submodules that are
known to contribute towards lipid overproduction
phenotypes - Fatty acid elongation pathway (white),
Glycerophospholipid biosynthesis (cyan), Sphingoli­
pid biosynthesis (green), Storage lipids (brown), Ste­
rols (gray) and Sterol esters (red). A-CoA, acetyl
coenzyme A; M-CoA, malonyl coenzyme A; F-CoA,
fatty acyl coenzyme A; DHAP, dihydroxyacetone
phosphate; Glyc-3-P, glycerol 3-phosphate; LPA,
lysophosphatidic acid; PA, phosphatidic acid; CDP-
DAG, cytidine diphosphate diacylglycerol; PS, phos­
phatidylserine; PE, phosphatidylethanolamine; PME,
phosphatidylmethylethanolamine; PDME, phosphati­
dyldimethylethanolamine; PC, phosphatidylcholine;
PI, phosphatidylinositiol; DAG, diacylglycerol; TAG,
triacylglycerol; VLCF-CoA, very long chain fatty acyl
coenzyme A; DHS, dihydrosphingosine; DHS-P,
dihydrosphingosine phosphate; FFA, free fatty acid;
Ethn-P, ethanolamine phosphate; F-ald, fatty alde­
hyde; IPC, inositolphosphorylceramide; MIPC, man­
nosyl inositolphosphorylceramide; M(IP)2C,
mannosyl-diinositolphosphoceramide. Solid lines
indicate the general direction of flux, dashed lines
indicate the presence of recycle reactions or regula­
tory interactions.
S. Mishra et al.
introducing single gene deletions in the reference strain BY4741 as well
as the 3 overproduction reference strains. The genes targeted belong to
the glycerophospholipid, sphingolipid, and fatty acyl transfer sub­
networks. Quantitative lipidomic data from the overproduction refer­
ence strains and the single gene deletion mutants constructed in these
strains resulted in a more comprehensive dataset to train the kinetic
model (Tables S4–S7). Parameter estimation was then performed with
this dataset using the procedure described in the Methods section above.
A multi-start approach was employed, and 1000 guess vectors
generated with gradient optimization starting from each of the guesses.
The initial conditions of observed variables were fixed to the measured
lipid values of the reference strain BY4741, thus reducing the number of
parameters to be estimated. From these optimization trajectories, the 10
best results (lowest objective function value) were collected to create a
population of models for further analysis. Goodness-of-fit plots were
constructed for this population of models to assess how close the model
matched experimental data. The goodness-of-fit plot for the lipid mea­
surements of reference strain BY4741 and the 3 constructed strains
(scFA-4, scFA-5 and scFA-7) are shown in Fig. 2. The mean for each lipid
class was calculated by integrating each of the 10 models from the
chosen population of models. As can be seen in the plot, most predicted
values closely match the actual measurements. The plot has an R2 value
of 0.963 and a mean squared error (MSE) of 1.43 × 10− 3.
3.3. Analysis of known mutants using the kinetic model
The kinetic model was first used to perform post-hoc analysis
comparing the strategies and outcomes presented in previous studies of
lipid-related metabolic engineering in S. cerevisiae reported in literature.
The model predicts a flux distribution of the lipid biosynthetic reactions
thus providing a possible reason for the success or failure of mutants
reported in literature. Briefly, we compared the performance of similar
mutations constructed in the two studies focusing on fatty alcohol
overproduction in S. cerevisiae, specifically the knockout of a tran­
scriptional regulator of lipid biosynthesis, OPI1 (d’Espaux et al., 2017;
Fig. 2. Goodness-of-fit plot of predictions from the population of models
plotted versus the lipid values for the reference strain BY4741 (red) as well as 3
platform strains, scFA-4 (green), scFA-5 (blue) and scFA-7 (yellow). Mean
values for each lipid class were calculated from 10 model predictions belonging
to the population of models. R2 and mean squared error (MSE) are marked on
the figure.
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Metabolic Engineering 75 (2023) 12–18
Feng et al., 2015). The two studies reported opposing effects on fatty
alcohol titers from the knockout of this genes. OPI1 is an inhibitor of the
INO2–INO4 heterodimer, known to activate various lipid biosynthetic
genes, while an increase in PA levels suppresses the effect of OPI1 by
tethering to it (Henry et al., 2012). In our model, we implemented the
action of OPI1 as indirect activation of lipid biosynthetic genes by PA, as
suggested elsewhere (Ferreira et al., 2018). Thus, modeling the
knockout of OPI1 simply involved removing the regulation terms by PA.
We first implemented the base strains reported in these two studies
and then enforced the effects of knockouts of OPI1 to observe the pre­
dicted flux distributions as well as fatty alcohol titers (Fig. 3). According
to the model, the success of OPI1 knockout in the study by Feng and
coworkers (24) could be traced to an increase in phospholipid biosyn­
thesis, which was accompanied by a commensurate increase in flux of
fatty acyl-CoA biosynthesis that could be converted to fatty alcohol. In
contrast, the model attributed the failure of the same strategy in the
study by d’Espaux and coworkers (24) to the lack of increase in fatty
acyl-CoA flux. The authors of the latter study employed the constitutive
TEF1 promoter to increase the flux in the fatty acyl-CoA biosynthetic
steps. This effectively decoupled expression of ACC1, FAS1 and FAS2
from any lipid regulation as the TEF1 promoter lacks the inositol-
sensitive upstream activating sequence (UASINO) characteristic to
many promoters in lipid metabolism that enables regulation at the gene
expression level (Henry et al., 2014). Thus, knockout of OPI1 led to an
increase in expression of other lipid biosynthetic genes without a similar
increase in fatty acyl-CoA flux. This imbalance in fluxes at the fatty
acyl-CoA node causes a bottleneck resulting in lowered fatty alcohol
titers.
3.4. Lipid model as a hypothesis generator: discovery of a futile cycle in
TAG biosynthesis
Next, the model was used to assist in designing mutants for fatty
alcohol overproduction. A strategy of accumulating triacylglycerol
(TAG) via knockouts in the scFA-7 (BY4741 acc1:PTEF1-ACC1, fas1:
PTEF1-FAS1, fas2: PTEF1-FAS2, X4: PTEF1-MmFAR1-TCYC1) over­
production platform strain (producing fatty alcohol) followed by chan­
neling the accumulated carbon towards fatty alcohol was implemented.
Based on simulation predictions, we constructed various knockout mu­
tants in the scFA-7 strain aimed at increasing the triacylglycerol (TAG)
amounts in the biomass. Knockout of the genes OPI3 and INO1 in scFA-7
showed the highest increase in TAG accumulation when normalized by
OD600 units (Supplementary Fig. S1). The TAG accumulating mutants
were then combined with overexpression of FAA1, FAA4 and TGL3
Fig. 3. Prediction of fatty alcohol titers on implementation of OPI1 knockouts
in each of the production strains described in either Feng et al. (Feng et al.,
2015) or d’Espaux et al., 2017. Prediction mean and standard deviations were
calculated from simulating the population of models.
S. Mishra et al. Metabolic Engineering 75 (2023) 12–18

genes, which are expected to increase the flux of fatty acyl chain hy­
drolysis from TAG and convert them back to fatty acyl-CoA. However,
this had no noticeable effect on the fatty alcohol titers, and on further
inspection, the TAG levels showed no appreciable decrease in their
levels (Supplementary Fig. S1). On the other hand, the triple over­
expression strain of FAA1, FAA4 and TGL3 showed an increase in the
levels of diacylglycerol (DAG) and ergosterol ester (Supplementary
Fig. S2).
The lack of noticeable change in TAG levels were inspected through
in silico analysis using the trained kinetic models. The predicted flux
values from the kinetic model showed a concomitant increase in both
the in and out flux of TAG pools, i.e. in the TAG synthesis and TAG
degradation rates. These flux trends suggested the action of a futile cycle
in TAG biosynthesis. A futile cycle is said to exist when a reaction con­
verting a substrate to a product is counteracted by a reaction in the
opposite direction replenishing the substrate, leading to a net con­
sumption of ATP without any change in metabolite levels (Stephano­
poulos et al., 1998).
Experimental validation of the increased flux within the TAG
biosynthesis – degradation cycle was performed by a labeled isotope
experiment with dynamic sampling. At 24 h of growth in SC media, the
control and mutant strains were washed and resuspended in SC media
containing 2% 13C6 – glucose and the cultures sampled at timepoints of
1, 2, 3, 4 and 8 h. Lipidomic analysis of the intracellular contents
coupled with HPLC analysis of remaining 13C6 – glucose in the media
were used to relate consumed carbon to produced TAGs. The biomass-
normalized amounts of 13C TAG accumulated showed a roughly linear
trend in both strains from 1 to 4 h of sampling. The absolute rate of 13C
incorporation into TAG pools was used as a proxy for rate of synthesis of
TAG and contrasted with the biomass-predicted flux of TAG requirement
(Fig. 4A). Specific growth rates of both the control strain and the mutant
strain, required for the predicted TAG requirement, were estimated from
the exponential phase of the growth curves (Fig. 4B). As can be seen in
Fig. 4C, the mutant strain showed a significant increase in actual TAG
incorporation over the predicted TAG requirement, whereas the control
strain showed nearly equally matched rates. The mismatch in the mutant
strain incorporation rates indicates the presence of a futile cycle in TAG
synthesis-degradation, suggesting that the mutations in this strain may
have weakened the control architecture implemented by the host strain
to prevent futile cycles in TAG biosynthesis.
A single exponential enrichment model (Buescher et al., 2015) was
assumed for all the measured lipids within the TAG
biosynthesis-degradation cycle, namely PA, DAG and TAG. The enrich­
ment data along with pool size and time of sampling was used to
calculate the flux of 13C carbon entering the pools. The calculated flux
values were upregulated in all 3 lipids in the mutant strain compared to
the control (Fig. 5). The concomitant increase of flux in all the major
lipids within the TAG biosynthesis cycle establishes the presence of a
futile cycle in the mutant around TAG biosynthesis and degradation.
Fig. 4. A) Time series plot of incorporation of 13C into the TAG pools of both
4. Discussion control and mutant strains. B) Growth curves of both control and mutant strains
during the 13C labeling experiment, where labeled substrate was introduced at
In this work, we developed a lipid kinetic model that was designed t = 0 h. C) Comparison of TAG requirements as predicted from biomass cal­
and trained to guide metabolic engineering of S. cerevisiae for lipid culations and actual synthesis of TAG, as estimated by rate of 13C incorporation.
overproduction. The model was constructed to include all essential The comparison shows a clear increase in TAG incorporation in the mutant
submodules of the lipid metabolic network of S. cerevisiae that are strain over its predicted requirements.
known to contribute towards lipid overproduction phenotypes. The
training dataset for the model included intracellular and extracellular parameters estimated in this study specifically capture the phenotypes of
lipidomic profiles of the reference S. cerevisiae strain BY4741 as well as lipid overproduction (Alvarez-Vasquez et al., 2005, 2011; Savoglidis
mutants constructed to have lipid overproduction phenotypes. This et al., 2016). This enabled a closer scrutiny of flux through the lipid
enabled the model to both capture the trends of lipidomic shifts occur­ metabolic map when the reactions carried a higher amount of flux for
ring in overproduction mutants and predict the necessary genetic ma­ overproduction. Specifically, the model was trained on data from the
nipulations required for further overproduction mutants to be reference strain BY4741 as well strains that overproduce free fatty acids,
constructed. The use of overproduction strains and perturbation muta­ fatty acyl-CoA and fatty alcohols.
tions constructed on these reference strains sets the lipid kinetic model Designing a training dataset that includes single gene knockout
presented here apart from earlier published models as kinetic mutants constructed on the original reference strain BY4741 and the

16
S. Mishra et al.
three overproduction reference strains assisted in estimating parameters
for a model that closely matched the training dataset (Fig. 2). Methods
such as Design of Experiments can be successfully employed in the
future to design optimal perturbation experiments that expand the
prediction space of a kinetic model. As demonstrated in this study, the
model can also be used to interpret and hypothesize mechanistic reasons
for the success or failure of mutant designs in metabolic engineering
literature. Model simulations comparing the lipid profiles of knockouts
of OPI1, a transcriptional regulator of lipid biosynthesis, provided a
reasonable explanation for the discrepancies noticed in their impact on
fatty alcohol titers (d’Espaux et al., 2017; Feng et al., 2015).
The presence of cycles in the lipid network makes the emergence of
futile cycles a real possibility to contend with during lipid metabolic
engineering. Combining insights from in silico analysis of the model and
13
C labeling data, we studied the emergence of a futile cycle in the tri­
acylglycerol biosynthesis and degradation pathways within a mutant
constructed for fatty alcohol overproduction. In humans, studies have
reported futile cycles around TAG biosynthesis that were activated
under interventions such as drug treatment (Guan et al., 2002). Even in
S. cerevisiae, a futile cycle with TAG biosynthesis has been reported
under various conditions deviating from wild-type behavior, such as
mutations in the enzyme glycerol-3-phosphate acyltransferase (GPAT)
which catalyzes the first committed reaction for TAG synthesis (Kiegerl
et al., 2019), or the removal of an endoplasmic reticulum membrane
protein required for efficient utilization of diacylglycerol (Markgraf
et al., 2014). In silico methods have also been developed to identify futile
cycles in genome-scale models (Gebauer et al., 2012). To investigate a
potential TAG futile cycle, we combined an in silico analysis with 13C
labeling experiments. We utilized kinetic model simulations to formu­
late a hypothesis for the cycling of carbon flux in the TAG biosynthesis
and degradation pathways. Higher incorporation rates of 13C in TAG and
precursor intermediates demonstrated the existence of such a cycle
(Figs. 4 and 5). In contrast with previously reported TAG futile cycles,
the cycle in this study relied on an upregulated TAG degradation and
FFA activation pathway. Our findings demonstrate the fine balance that
exists between TAG synthesis and degradation pathways and suggest the
need for more strategies that can effectively channel carbon from stor­
age lipids towards valuable products without creating an imbalanced
system. Insights into the TAG synthesis-degradation balance may enable
design of future strains presenting oleaginous phenotypes.
In conclusion, we developed a kinetic model of lipid metabolism in
S. cerevisiae that was trained on phenotypes relevant to metabolic
17
Metabolic Engineering 75 (2023) 12–18
Fig. 5. A) A reduced map of the TAG biosynthesis
cycle which is initiated by the successive acylation of
glycerol 3-P into LPA and PA. PA is then dephos­
phorylated to DAG, and DAG is finally acylated to
synthesize TAG. The action of lipases convert TAG
back to DAG along with the release of FFA. The
activation of FFA to fatty acyl-CoA completes the
cycle of reactions in TAG biosynthesis. To obtain es­
timates of flux of 13C incorporation into different
lipids in the TAG biosynthesis cycle, a single expo­
nential enrichment model was used. The flux values
of three lipid classes are as follows, B) PA, C) DAG,
and D) TAG.
engineering. The utility of the model was demonstrated while designing
mutants for fatty alcohol overproduction, during which a futile cycle
was discovered in the TAG biosynthesis pathways. The ability of the
model to explain successful and unsuccessful mutant designs in meta­
bolic engineering literature also showcases its potential in assisting the
design of future mutant strains for overproduction of lipid-based target
chemicals.
Author contributions
Shekhar Mishra: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Software, Writing – Original draft,
Visualization.
Ziyu Wang: Investigation.
Michael J. Volk: Investigation.
Huimin Zhao: Conceptualization, Writing – Original draft.
Data availability
Data will be made available on request.
Acknowledgements
The authors wish to thank Dr. Jay Keasling for generously providing
the MmFAR1 gene. The authors also thank Dr. Yihui Shen for helpful
discussions on 13C data analysis. The authors wish to thank Dr. Alex­
ander Ulanov and Dr. Michael La Frano at the Metabolomics Center of
the Roy J. Carver Biotechnology Center at the University of Illinois,
Urbana-Champaign. This work was supported by the U.S. Department of
Energy (DE-SC0018260) (H.Z.).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ymben.2022.11.003.
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