The fatty acid and lipid consumption in the world is increasing day by day, as lipids are required
in a wide range of applications including personal care, home care, pharmaceuticals, biofuels,
food additives, etc. With the world’s population growing, the demand for lipid-based products is
also increasing dramatically (Abdelmoez & Mustafa, 2014).
Lipids are the building blocks of cellular membranes and they also have many other essential
biological roles in the cell. They serve the cell as energy storage and they also play a significant
role in cell signaling pathways (Olzmann & Carvalho, 2019). Neutral lipids are a subgroup of
lipids that consist of hydrophobic molecules without charged groups. Triacylglycerols (TAGs)
and sterol esters (SE) comprise a major class of neutral lipids that are stored in lipid droplets
(LDs). LDs are dynamic storage organelles emerging from the endoplasmic reticulum (ER) and
they stay connected to ER throughout their biogenesis and life cycle. The hydrophobic core of
LD consists of TAGs that are surrounded by several layers of SEs. The core is enclosed by a
phospholipid monolayer covered by perilipin proteins. LDs have contact sites with other cellular
organelles, such as Golgi apparatus and mitochondria. These contacts facilitate the exchange of
lipids, ions and metabolites (Athenstaedt, 2010).
Storage lipids in the form of TAGs are known to be one of the key targets for biotechnological
product development (Schörken & Kempers, 2009).
Microbial production is more favorable to the environment when compared to the conventional
ways. This has resulted in bioproduction becoming an attention-grabbing field for manufacturers
as a biosustainable alternative (Abdelmoez & Mustafa, 2014).
In SPARKLE, we chose Saccharomyces cerevisiae as a cell factory for TAG production as it is a
thoroughly studied model organism that is convenient to work with. Unlike some other species
of yeasts, S. cerevisiae has a low intrinsic ability for producing lipids (Johnson et al., 1972).
TAG content makes up around 1% of budding yeast cell’s dry weight. However, due to well-
developed protocols for genetic modification of S. cerevisiae, the metabolic pathways for lipid
production can be manipulated to enhance the level of TAG in yeast (Ferreira et al., 2018).
Additionally, S. cerevisiae is known to be a robust species that tolerates extreme fermentation
conditions (Zhou et al., 2016).
In yeast, fatty acid biosynthesis takes place in the cytosol and mitochondria. In our project, we were dealing with the cytosolic synthesis of FAs and further TAG assembly in the ER. We will not discuss here FA production in mitochondria. FAs are synthesized de novo in the cytosol in a series of reactions that convert the precursor malonyl‐CoA to long-chain fatty acids (i.e. fatty acids that contain more than 14 carbon atoms). . The first step of the conversion of acetyl‐CoA to fatty acids is its carboxylation to
malonyl‐CoA, a reaction catalyzed by the ACC1‐encoded acetyl‐CoA carboxylase. (Fakas,
2017) This is a rate-limiting step in fatty acid synthesis. (Sheng & Feng, 2015)
In de novo fatty acid biosynthesis, Fatty Acid Synthase (FAS) uses the generated malonyl-CoA
as a substrate and NADPH as a reducing agent (Ma et al., 2019). Each step of fatty acid elongation by two carbon units requires two NADPH molecules, which are produced in the cytosol of S. cerevisiae in the oxidative phase of the pentose phosphate pathway (PPP). The first reaction of PPP is catalyzed by glucose-6-phosphate dehydrogenase (encoded by the ZWF1 gene) (He et al., 2018; Sheng & Feng, 2015). During the first phase of PPP, when a hexose sugar is oxidized, two carbons are lost in the form of CO2. This leads to a decrease in theoretical carbon yield.
Further elongation and desaturation of the de novo synthesized FA occurs in the ER and results in the formation of very-long-chain fatty acids (usually, longer than C18). The majority of fatty acids in S. cerevisiae are monounsaturated palmitoleic (C16:1) and oleic (C18:1) acids, followed by saturated palmitic (C16:0) and stearic (C18:0) acids. The
resulting fatty acids participate in phosphatidate biosynthesis. The end product of this pathway is
phosphatidic acid (PA), which upon dephosphorylation by phosphatidic acid phosphatase (PAP,
encoded by PAH1) yields diacylglycerol (DAG). The final and the only committed step of TAG
biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT, encoded by
DGA1), which converts DAGs into TAGs.
Although TAG biosynthesis in S. cerevisiae occurs in the ER, the accumulation of TAG depends
on de novo fatty acid biosynthesis which begins in the cytosol, where Acc1 is localized. In yeast, the activity of Acc1 is post-translationally regulated by Snf1 protein kinase. It has
been shown that relieving the negative regulation of Acc1 activity by mutating the Snf1
phosphorylation sites increases the malonyl-CoA supply for fatty acid synthesis (Sheng & Feng,
In order to design an efficient and versatile platform for lipid production, we focused on
removing the major bottlenecks and maximizing the carbon flux through the lipid synthesis
pathway. Upon designing our project, we adopted the “Push, Pull, and Protect” strategy that was successfully employed in plants. It involves optimizing the flux of carbon into TAG (the storage form of
lipids) by expressing lipid-synthesizing enzymes to increase the fatty acid synthesis (Push),
increasing TAG assembly (Pull), and downregulating lipid turnover (Protect). (Vanhercke et al.,
We aim to increase fatty acid synthesis by two approaches: first, increasing the precursor supply,
and secondly, providing the cofactor NADPH without burdening the central carbon metabolism.
It has been shown that one of the bottlenecks of producing fatty acids in yeast is the tight
posttranslational regulation of Acc1 activity by Snf1. In order to remove the feedback inhibition,
we aim to replace ACC1 with a mutated version ACC1S659A,S1157A,S686A which lacks the
phosphorylation sites targeted by kinase Snf1. These mutations have been shown to lead to an
increase in Acc1 activity and malonyl-CoA for fatty acid synthesis (Sheng & Feng, 2015).
As mentioned above, NADPH is a key cofactor in lipid biosynthesis. NADPH is also required
for other metabolic processes, such as amino acid and cholesterol biosynthesis. Therefore, fatty
acid metabolism and cell growth in general are greatly constrained by an insufficient supply of
NADPH in the cytosol. To address that, some studies have focused on improving the NADPH
supply in cytosol (de Jong et al., 2014; Z. Guo et al., 2011). However, increased NADPH
regeneration leads to a decrease in theoretical carbon yield. In our project, we aimed to decouple
NADPH generation from the central carbon metabolism to maximize the carbon flux towards
The most straightforward solution for carbon loss would be to eliminate the oxidative portion of
the PPP. Doing so would reduce carbon loss in the form of CO2 but would inevitably also lead to
decreased availability of cytosolic NADPH. An alternative supply for the cytosolic NADPH has
been suggested by Guo et al. (2018). The scientists have created a semiconductor biohybrid
system by functionalizing genetically engineered yeast cells with light-absorbing indium
phosphide (InP) nanoparticles.
The electrons photogenerated by surface-bound InP nanoparticles enable the regeneration of NADPH
from NADP+ without the oxidation of hexose sugar in PPP and the concomitant loss of CO2 (Guo et al., 2018). The deletion of Zwf1, a gene coding for glucose-6-phosphate dehydrogenase
which catalyzes the first step of PPP, disrupts the oxidative portion of the pathway. Cells bearing the mutation have a
decreased ability to regenerate cytosolic NADPH which can be complemented by electron
donation from an illuminated semiconductor. This approach results in 2 outcomes: NADPH
is overproduced and carbon flux is enhanced towards fatty acid biosynthesis.
There are two reasons for using InP as the photosensitizer. First, the optical and electronic
properties of nanoscale InP enable efficient absorption of a large fraction of solar spectrum (Guo et al., 2018). Secondly, InP is by far one of the most biocompatible and sustainable
semiconductors in biohybrid engineering. Taken together, these two factors make it a suitable
material for solar-driven biocatalysis.
It has been shown that overexpression of PAH1 (converts PA to DAG) and DGA1 (converts
DAG to TAG) (Fig.1) leads to an increase in accumulation of TAG in S. cerevisiae (Teixeira et
al., 2018). However, simple overproduction of lipid synthesizing enzymes impedes cell growth.
More specifically, endogenous pathways that are essential for cell growth may compete with
lipid biosynthesis pathway, especially following the exhaustion of carbon source in the culture
medium (Papanikolaou & Aggelis, 2011; Zhao et al., 2018). This, in turn, leads to a decreased
biomass and reduced production efficiency. To address this, scientists frequently use inducible
systems to uncouple the competing pathways (Zhao et al., 2018). This approach enables to
separate a growth phase, when additional lipid biosynthesis genes are not expressed, and a
production phase, when carbon flux through the lipid biosynthesis pathway is maximized.
In our project, we have implemented the VP-EL222 optogenetic system, which utilizes a light-sensitive transcription factor VP-EL222 from Erythrobacter litoralis. With this system, each
gene (PAH1, DGA1) is placed under the control of a VP-EL222-regulated promoter. In the
absence of blue light, the genes which would otherwise inhibit biomass growth are not
expressed. However, once the cells are exposed to blue light, the overexpression of lipid
metabolism genes is switched on and the production phase starts, in which carbon accumulates in
the form of lipids.
There are a number of different inducible expression systems, where an exogenous effector
molecule governs both the onset and the extent of transcription of a specific promoter. However, once
added to the medium and entered the cell, such molecules tend to linger, as they can not be
cleared rapidly and even could contaminate the final product. Additionally, some inducer
molecules are expensive and are incompatible with some nutrients in media. Optogenetic tools,
on the other hand, do not require changes in the medium and therefore can be applied and
removed instantly. Moreover, optogenetic tools have different wavelength specifications, which
allows for additional layers of control in biological systems.
Since lipids have an important structural and functional role in the cell, TAGs accumulated in
LDs can be hydrolyzed and released upon requirement. Unlike some cells that utilize their lipid
reserves mainly as a source of chemical energy, S. cerevisiae uses these degradation products as
building blocks for membrane lipid synthesis (Czabany et al., 2007). Still, downregulation of
lipid turnover is important to achieve high lipid titers.
It has been shown that knockout of TAG-hydrolysis pathways can improve TAG accumulation
(Ferreira et al., 2018). Therefore, our final strain will lack the main lipase genes TGL3, TGL4
LDs sequester TAGs, which is necessary for their stabilization and to prevent lipotoxicity
(Olzmann & Carvalho, 2019). To further protect the accumulated TAGs, we aim to promote LD
assembly by ectopic expression of human gene PLIN3. PLIN3 encodes for a perilipin, which
covers the phospholipid monolayer of LDs. Expression of PLIN3 in yeast cells has been
demonstrated to considerably increase TAG content. We will construct an inducible PLIN3
expression cassette, that will be activated by light-inducible VP-EL222 transcription factor
together with the enzymes promoting TAG synthesis, Pah1 and Dga1.
Downstream processing is an integral part of the production process and has significant costs
associated with it. It has been estimated that product extraction and purification often takes 85%
of the total costs in biomanufacturing process. In addition to being energy-intensive, lipid
extraction involves the use of high amounts of toxic solvents that require cell wall disruption for
effective extraction (Yu et al., 2015). Moreover, when the lipids are manufactured for food
industry, toxic chemicals should be avoided. Taken together, the cost and the complexity of
downstream processing are two limiting factors that make bioproduction less competitive in
comparison to chemical synthesis (Reneberg, Berkling and Loroch, 2016).
An easier and less expensive process of lipid recovery, for example, if the lipids were secreted
directly to the medium, would make lipid bioproduction more competitive (Vasconcelos et al.,
2019). Lipid extraction from wet biomass would significantly reduce the energy spent
dewatering the cell biomass. However, the available technologies are far from being
commercialized (Dong et al., 2016). Our team has previously shown that induced expression of
bacterial glucanases has a potential to cause cell lysis. In that project the glucanases were secreted
from the cells. However, the efficiency of autolysis could be improved by increasing the local
concentration of glucanases in the cell wall. The bacterial glucanases could potentially be
anchored to yeast cell wall by fusion with GPI-anchored cell wall proteins (Inokuma et al.,
2020). In order to ease lipid extraction from yeast cells, we aimed to improve the inducible yeast
autolysis by overexpressing β-1,3-glucan laminaripentao-hydrolase (Glc1) and endo-1,3-β-
glucanase from Cellulosimicrobium cellulans (BBa_K2711000) fused to GPI-anchors.
Chen, X., Yang, X., Shen, Y., Hou, J., and Bao, X. (2018). Screening Phosphorylation Site
Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoA Sensor to Improve
Malonyl-CoA-Derived Product. Frontiers in Microbiology, 9, 47.
de Jong, B.W., Shi, S., Siewers, V., and Nielsen, J. (2014). Improved production of fatty acid
ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation
pathway and expression of the heterologous phosphoketolase pathway. Microbial Cell
Factories, 13(1), 39.
Dong, T., Knoshaug, E.P., Pienkos, P.T., and Laurens, L.M.L. (2016). Lipid recovery from wet
oleaginous microbial biomass for biofuel production: A critical review. Applied Energy,
Fakas, S. (2017). Lipid biosynthesis in yeasts: A comparison of the lipid biosynthetic pathway
between the model nonoleaginous yeast Saccharomyces cerevisiae and the model
oleaginous yeast Yarrowia lipolytica. Engineering in Life Sciences, 17(3), 292–302.
Ferreira, R., Teixeira, P.G., Gossing, M., David, F., Siewers, V., and Nielsen, J. (2018). Metabolic
engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols. Metabolic
Engineering Communications, 6, 22–27.
Guo, J., Suástegui, M., Sakimoto, K.K., Moody, V.M., Xiao, G., Nocera, D.G., and Joshi, N.S.
(2018). Light-driven fine chemical production in yeast biohybrids. Science, 362(6416), 813
LP – 816.
Guo, Z., Zhang, L., Ding, Z., and Shi, G. (2011). Minimization of glycerol synthesis in industrial
ethanol yeast without influencing its fermentation performance. Metabolic Engineering,
He, Q., Yang, Y., Yang, S., Donohoe, B.S., Van Wychen, S., Zhang, M., Himmel, M.E., and
Knoshaug, E. P. (2018). Oleaginicity of the yeast strain Saccharomyces cerevisiae D5A.
Biotechnology for Biofuels, 11, 258.
Inokuma, K., Kurono, H., den Haan, R., van Zyl, W.H., Hasunuma, T., and Kondo, A. (2020).
Novel strategy for anchorage position control of Glycosylphosphatidylinositol(GPI)-attached proteins in the yeast cell wall
using different GPI-anchoring domains. Metabolic Engineering, 57, 110–117.
Ma, T., Shi, B., Ye, Z., Li, X., Liu, M., Chen, Y., Xia, J., Nielsen, J., Deng, Z., and Liu, T. (2018). Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metabolic Engineering, 52.
Papanikolaou, S., and Aggelis, G. (2011). Lipids of oleaginous yeasts. Part I: Biochemistry of
single cell oil production. European Journal of Lipid Science and Technology, 113(8),
Reneberg, R., Berkling, V., and Loroch, V. (2016). Biotechnology for Beginners 2nd Edition (2nd ed.). Academic Press.
Sheng, J., and Feng, X. (2015). Metabolic engineering of yeast to produce fatty acid-derived
biofuels: bottlenecks and solutions . In Frontiers in Microbiology (Vol. 6, p. 554).
Vanhercke, T., El Tahchy, A., Liu, Q., Zhou, X.-R., Shrestha, P., Divi, U.K., Ral, J.-P.,
Mansour, M. P., Nichols, P. D., James, C. N., Horn, P.J., Chapman, K. D., Beaudoin, F.,
Ruiz-López, N., Larkin, P.J., de Feyter, R.C., Singh, S.P., and Petrie, J.R. (2014).
Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol
yields from plant leaves. Plant Biotechnology Journal, 12(2), 231–239.
Vasconcelos, B., Teixeira, J.C., Dragone, G., and Teixeira, J. A. (2019). Oleaginous yeasts for
sustainable lipid production—from biodiesel to surf boards, a wide range of “green”
applications. Applied Microbiology and Biotechnology, 103(9), 3651–3667.
Yu, X., Dong, T., Zheng, Y., Miao, C., and Chen, S. (2015). Investigations on cell disruption of
oleaginous microorganisms: Hydrochloric acid digestion is an effective method for lipid
extraction. European Journal of Lipid Science and Technology, 117(5), 730–737.
Zhao, E.M., Zhang, Y., Mehl, J., Park, H., Lalwani, M.A., Toettcher, J.E., and Avalos, J.L.
(2018). Optogenetic regulation of engineered cellular metabolism for microbial chemical
production. Nature, 555(7698), 683–687.
Abdelmoez, W., & Mustafa, A. (2014). Oleochemical industry future through biotechnology.
Journal of Oleo Science, 63(6), 545—554.
Athenstaedt, K. (2010). Neutral Lipids in Yeast: Synthesis, Storage and Degradation. Handbook
of Hydrocarbon and Lipid Microbiology, 471–480.
Schörken, U., and Kempers, P. (2009). Lipid biotechnology: Industrially relevant production processes.
European Journal of Lipid Science and Technology, 111, 627–645.
Johnson, B., Nelson, S.J., and Brown, C.M. (1972). Influence of glucose concentration on the
physiology and lipid composition of some yeasts. Antonie van Leeuwenhoek, 38(1),
Olzmann, J.A., and Carvalho, P. (2019). Dynamics and functions of lipid droplets. Nature
Reviews Molecular Cell Biology, 20(3), 137–155.
Zhou, Y.J., Buijs, N.A., Zhu, Z., Qin, J., Siewers, V., and Nielsen, J. (2016). Production of fatty
acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nature
Communications, 7(1), 11709.
The needs of an intensively growing human population lead to increasing demands in lipid-derived compounds widely used for the production of a variety of products: from everyday items to biofuels. In 2019 the oleochemical market was valued at 20.1 billion USD (Grand View Research, 2020). However, gradual depletion of non-renewable energy sources, enhanced emission of greenhouse gases, which are considered to be the main cause of global warming, and growing amount of industrial waste has caused a need to find more sustainable sources of lipid-derived compounds.
Oleaginous plants such as oil palms are widely used as producers of oil-derived compounds. However, they have a long life cycle and oil palm agriculture is associated with deforestation, and loss of biodiversity (Vijay et al., 2016). Microbial bioproduction is thought to be the future for the oleochemical industry as it has the potential to solve the problems mentioned above.
This year, our team decided to contribute to the generation of an alternative source for lipid production: we created a yeast cell factory that performs light-dependent lipid droplet biosynthesis. The cell factory is based on genetically modified Saccharomyces cerevisiae coated with light-absorbing nanoparticles (Guo et al. 2018). In our cell factory, light plays the role of an inductor that switches the metabolism to start lipid production and provides an energy source to drive the synthesis. By this means, we can split the growth and production phases to make the cell factory more efficient. Further, this approach can reduce production-associated cost as light is a relatively cheap source of energy, and minimize waste generation in comparison to chemical synthesis.
Grand View Research (2020). Oleochemicals Market Size, Share | Industry Report, 2020-2027. Available at: https://www.grandviewresearch.com/industry-analysis/oleochemicals-industry.
(Accessed: 23rd October 2020)
Guo, J., Suástegui, M., Sakimoto, K., Moody, V., Xiao, G., Nocera, D., and Joshi, N. (2018). Light-driven fine chemical production in yeast biohybrids. Science 362, 813-816.
Vijay, V., Pimm, S., Jenkins, C., and Smith, S. (2016). The Impacts of Oil Palm on Recent Deforestation and Biodiversity Loss. PLOS ONE 11, e0159668.