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¹iGEM Student Team Member, ²iGEM Team Avdisor, ³iGEM Team Instructor, ⁴Secondary PI, ⁵Primary PI, Institute of Technology, University of Tartu, Tartu, Estonia | ¹iGEM Student Team Member, ²iGEM Team Avdisor, ³iGEM Team Instructor, ⁴Secondary PI, ⁵Primary PI, Institute of Technology, University of Tartu, Tartu, Estonia | ||
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<h3>Abstract</h3> | <h3>Abstract</h3> | ||
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Yeasts have the potential to be used as cell factories to produce lipids (biodiesel, high-value lipids, etc.). However, bio-production is costly compared to chemical synthesis, as it is highly energy-consuming for the cell and product extraction is laborious. To increase competitiveness, we engineer yeast to accumulate high lipid levels by using light both as an inductor for metabolic switch and as an electron source. Further, yeast is designed to self-lyse after production. First, we introduce extra copies of lipid synthesizing enzymes controlled by light-inducible promoters. Next, we coat the cells with light-absorbing nanoparticles to enable the cells to use light as an electron source for NADPH formation – a critical cofactor for lipid synthesis. This leads to increased carbon flux to lipid production. To ease the product extraction, the cells are designed to autolyse by induction of cell wall degrading glucanases that are targeted to the cell wall via anchor proteins. | Yeasts have the potential to be used as cell factories to produce lipids (biodiesel, high-value lipids, etc.). However, bio-production is costly compared to chemical synthesis, as it is highly energy-consuming for the cell and product extraction is laborious. To increase competitiveness, we engineer yeast to accumulate high lipid levels by using light both as an inductor for metabolic switch and as an electron source. Further, yeast is designed to self-lyse after production. First, we introduce extra copies of lipid synthesizing enzymes controlled by light-inducible promoters. Next, we coat the cells with light-absorbing nanoparticles to enable the cells to use light as an electron source for NADPH formation – a critical cofactor for lipid synthesis. This leads to increased carbon flux to lipid production. To ease the product extraction, the cells are designed to autolyse by induction of cell wall degrading glucanases that are targeted to the cell wall via anchor proteins. | ||
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<div class="title"> Description </div> | <div class="title"> Description </div> | ||
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− | <div class="text"> With the world’s population growing, the global fatty acid and lipid consumption is also increasing dramatically, as these compounds are used in a wide range of applications (Abdelmoez & Mustafa, 2014). <br> | + | <div class="text"> |
+ | <img src="https://static.igem.org/mediawiki/2020/c/cd/T--Estonia_TUIT--Poster_Design_fig1.png"> | ||
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+ | With the world’s population growing, the global fatty acid and lipid consumption is also increasing dramatically, as these compounds are used in a wide range of applications (Abdelmoez & Mustafa, 2014). <br> | ||
Lipids have essential structural and biological roles in the cell, as they are the building blocks of cellular membranes, they serve the cell as energy storage and they participate in signaling pathways (Olzmann & Carvalho, 2019). <br> | Lipids have essential structural and biological roles in the cell, as they are the building blocks of cellular membranes, they serve the cell as energy storage and they participate in signaling pathways (Olzmann & Carvalho, 2019). <br> | ||
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). The hydrophobic core of LD consists of TAGs and is enclosed by a phospholipid monolayer covered by perilipin proteins (Athenstaedt, 2010). TAGs are one of the main targets for biotechnological product development. | 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). The hydrophobic core of LD consists of TAGs and is enclosed by a phospholipid monolayer covered by perilipin proteins (Athenstaedt, 2010). TAGs are one of the main targets for biotechnological product development. | ||
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In SPARKLE, we improve the cytosolic synthesis of fatty acids and further TAG assembly in the ER. To achieve this, we adopted the “Push, Pull, and Protect” strategy. We aimed to direct the carbon flux into TAG (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). | In SPARKLE, we improve the cytosolic synthesis of fatty acids and further TAG assembly in the ER. To achieve this, we adopted the “Push, Pull, and Protect” strategy. We aimed to direct the carbon flux into TAG (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). | ||
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We increased the fatty acid synthesis by two approaches. First, we set out to increase the supply of a critical precursor, malonyl-CoA. Acetyl-CoA carboxylase, encoded by AСС1 gene, synthesizes malonyl-CoA, which drives lipid synthesis. Mutating three phosphorylation sites (S659A, S686A, and S1157A) in Acc1 disrupts the downregulation of Acc1 activity and has been found to lead to an increase in malonyl-CoA abundance. <br> | We increased the fatty acid synthesis by two approaches. First, we set out to increase the supply of a critical precursor, malonyl-CoA. Acetyl-CoA carboxylase, encoded by AСС1 gene, synthesizes malonyl-CoA, which drives lipid synthesis. Mutating three phosphorylation sites (S659A, S686A, and S1157A) in Acc1 disrupts the downregulation of Acc1 activity and has been found to lead to an increase in malonyl-CoA abundance. <br> | ||
Secondly, we aimed to decouple NADPH generation from the central carbon metabolism to maximize the carbon flux towards lipid biosynthesis. NADPH is a critical cofactor in fatty acid synthesis and is normally provided by the pentose phosphate pathway (PPP). However, the regeneration of NADPH leads to loss of carbon atoms in the form of CO2. The deletion of ZWF1, a gene encoding for glucose-6-phosphate dehydrogenase that catalyzes the first step of PPP, disrupts the oxidative portion of the pathway. Cells bearing this deletion have decreased ability to regenerate cytosolic NADPH. In SPARKLE, this is compensated by a semiconductor biohybrid system, in which the yeast cells were coated with light-absorbing indium phosphide nanoparticles. These particles, when exposed to light, donate electrons to the cell for the regeneration of NADPH, providing a carbon-free bypass for NADPH regeneration. | Secondly, we aimed to decouple NADPH generation from the central carbon metabolism to maximize the carbon flux towards lipid biosynthesis. NADPH is a critical cofactor in fatty acid synthesis and is normally provided by the pentose phosphate pathway (PPP). However, the regeneration of NADPH leads to loss of carbon atoms in the form of CO2. The deletion of ZWF1, a gene encoding for glucose-6-phosphate dehydrogenase that catalyzes the first step of PPP, disrupts the oxidative portion of the pathway. Cells bearing this deletion have decreased ability to regenerate cytosolic NADPH. In SPARKLE, this is compensated by a semiconductor biohybrid system, in which the yeast cells were coated with light-absorbing indium phosphide nanoparticles. These particles, when exposed to light, donate electrons to the cell for the regeneration of NADPH, providing a carbon-free bypass for NADPH regeneration. |
Revision as of 04:16, 11 November 2020