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, 2015).
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., 2014)
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 lipid biosynthesis. 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 and TGL5.
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.
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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.
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