Team:Estonia TUIT/Poster

SPARKLE: Solar-Powered ARtificially Knitted Lipid Enclosures Presented by Team Estonia_TUIT

Alissa Agerova¹, Avishan Aghayari¹, Amina Aliyeva¹, Nihat Aliyev, Norin Bhatti¹, Turan Badalli¹, Irina Borovko¹, Nadezhda Chulkova¹, Dmytro Fedorenko¹, Klāvs Jermakovs¹, Tatyana Kan¹, Valida Kazimova¹, Viacheslav Kiselev¹, Gleb Kovalev¹, Valeria Leoshko¹, Mykhailo Lytvynenko¹, Anna Makhotina¹, Dags Macs¹, Glib Manaiev¹, Frida Matiyevskaya¹, Mark Merzlikin¹, Juli Mukhadze¹, Alar Okas¹, Johanna Olesk¹, Aleksandra Panfilova¹, Aleksandrs Rebriks¹, Davit Rizhinashvili¹, Ekaterina Sedykh¹, Jhalak Sethi¹, Aleksandra Shabanova¹, Muazzamkhon Yusupova¹, Maksym Zarodniuk¹, Nastassia Shtaida², Mihkel Örd², Artemi Maljavin³, Ilona Faustova⁴, Mart Loog⁵
¹iGEM Student Team Member, ²iGEM Team Avdisor, ³iGEM Team Instructor, ⁴Secondary PI, ⁵Primary PI, Institute of Technology, University of Tartu, Tartu, Estonia

Abstract

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.

Goals and Inspiration

Lipid-derived products are used in a wide range of applications, and their consumption in the world is increasing rapidly (Abdelmoez & Mustafa, 2014). Our project aims to develop a cell factory for lipid production and thus provide a sustainable alternative to plant-, animal- and chemically derived lipid sources.

We were inspired by a “Push, Pull, and Protect” strategy that has successfully worked in plants (Vanhercke et al., 2014). It involves directing the carbon flux to TAG by expressing lipid-synthesizing enzymes that increase the fatty acid synthesis (Push), by increasing TAG assembly (Pull), and by down-regulating lipid turnover (Protect).

In our project, we set out to improve lipid production by four main approaches:

1. Increasing the precursor malonyl-CoA supply by mutating phosphorylation sites that would downregulate Acc1 activity.
2. Reducing carbon loss during NADPH regeneration by knocking out ZWF1 gene and restoring NADPH regeneration capability by coating the cells with semiconductor nanoparticles.
3. Improving lipid accumulation capacity of the cell by overexpressing PAP and DGAT enzymes that catalyze the final steps of TAG assembly as well as improving lipid droplet stability with the help of human perilipin Plin3.
4. Protecting the accumulated TAGs from hydrolysis by deleting TGL3, TGL4 and TGL5 genes which encode for TAG lipases.


We also aimed to ease lipid extraction from yeast cells. For this, we set out to improve the inducible yeast autolysis by fusing the cell-wall-degrading bacterial glucanases with cell wall anchoring domains to increase their local concentration.

Our Goals
1) To improve lipid accumulation in yeast with the aim to use it as a cell factory for TAG production.

2) To enable the yeast to use light as an electron source by coating the cells with nanoparticles to increase the carbon yield in lipid production.

3) To develop a cell wall autolysis method to ease the extraction of the accumulated lipids.

REFERENCES:

Abdelmoez, W., & Mustafa, A. (2014). Oleochemical industry future through biotechnology. Journal of Oleo Science, 63(6), 545—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.

Description

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).
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).
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. We chose Saccharomyces cerevisiae as a host for a cell factory for TAG production as it is a thoroughly studied model organism that can be conveniently engineered to achieve new qualities.

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).

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.
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.

To increase TAG assembly, we worked on the overexpression of PAH1 and DGA1 that encode for PAP and DGAT enzymes. By leading to DAG and TAG formation, these enzymes catalyze the final steps of TAG synthesis. Upon exposure to light, these genes are expressed at high levels in our strain, leading to an increase in TAG formation.
Moreover, to protect the accumulated TAGs from hydrolysis, we deleted TGL3, TGL4 and TGL5 genes, which encode for TAG lipases that hydrolyze the lipids. To further stabilize and protect the accumulated TAGs, we introduced a human perilipin gene PLIN3, also under the light-activated promoter, into our yeast.



Finally, to ease the extraction of lipids, glucanases that degrade the yeast cell wall could be targeted to the cell wall by anchoring proteins. Induction of these glucanases would lead to the breakdown of the cell wall and release of product.

REFRENCES:

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.

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, 13(1), 49–59.

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.

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.

Olzmann, J.A., and Carvalho, P. (2019). Dynamics and functions of lipid droplets. Nature Reviews Molecular Cell Biology, 20(3), 137–155.

Design

We aimed to improve three stages of the lipid bio-production process: biomass growth, product synthesis and extraction. During the first stage, we expose our strain to red light to enable our indium phosphide biohybrid strain to regenerate NADPH without the loss of carbon (Guo et al., 2018).
Following biomass accumulation, the lipid production is initiated by a metabolic switch when the overexpression of PAH1, DGA1 and PLIN3 is toggled on by exposure to blue light.
Extraction is achieved by implementing our team’s 2019 project, where we showed that expression of bacterial glucanases allows cell autolysis and easier extraction of the product.

Hardware

To test our engineered yeast strain we designed and built the bioreactor "SANYA". It performs the following functions:
•Maintaining a constant temperature of 30 °C;
•Keeping controllable illumination conditions: light with adjustable intensity at 465 and 625 nm wavelengths;
•Mixing the culture with the speed 300-500 rpm for equal distribution of light and supplementation;
•Keeping yeast isolated from the environment.
Our bioreaction in action:

Decreasing TAG turnover by lipase deletions

We made different deletions of major TAG lipases TGL3, TGL4 and TGL5 and analyzed the effect of these modifications on the lipid levels. We found that deletion of TAG lipases results in a considerable increase in lipid levels, with the triple deletion having the greatest effect (Fig 1).



Figure 1. Mean Nile red fluorescence intensities in single cells from the indicated strains. The bars show median and its 95% confidence intervals. ****, **, and ns note p-values <0.0001, <0.01, and >0.05, respectively, of pairwise comparisons with wild-type using Mann-Whitney U test.

Perilipin expression promotes lipid accumulation

Perilipins promote lipid droplet assembly. To take use of this, we introduced a construct where perilipin Plin3 was expressed from a light-inducible promoter. We found that when grown under light, these cells accumulated substantially higher concentration of lipids (Fig 2), presumably because the induced Plin3 stabilizes TAGs by supporting lipid droplet formation.



Figure 2. Microscopy images of exemplary cells showing the accumulation of lipids in LDs. (B) Plot showing the average fluorescence intensities of Nile red staining. The bars show median values and its 95% confidence intervals. **** indicates p-value <0.0001 in pairwise comparison by Mann-Whitney U test.

Yeast biohybrids use light to drive lipid synthesis

We used nanoparticles to give our production strain a new quality – the ability to use light as an energy source to drive lipid synthesis (Fig 3). For that, we developed a bioinorganic hybrid platform where a disruption in the oxidative portion of the pentose phosphate pathway by ZWF1 deletion, which renders the cell unable to regenerate NADPH, is complemented by electron donation from an illuminated semiconductor. The experiments show that these biohybrids accumulate higher levels of lipids when grown under light, indicating that they are using light to support the lipid production.



Figure 3. (A) Microscopy images of exemplary cells showing the accumulation of lipids in LDs detected by Nile red staining. (B) Plot showing the average fluorescence intensities of Nile red staining. The bars show median values and its 95% confidence intervals. **** indicates p-value <0.0001 in pair-wise comparison by Mann-Whitney U test.

Modeling

We used 2 approaches to model the lipid production: an agent-based stochastic approach for a cell-scale model and an ODE approach for a culture-scale model.

First, we used an agent-based stochastic approach to model the effects of PAH1 and DGA1 overexpression combined with the reduction of TAG lipase activity. The ‘agents’ of the model are enzymatic reactions, for which the parameters can be changed. Overexpression of PAH1 and DGA1 led to an increased activity of PAP and DGAT enzymes that are responsible for the final steps in TAG synthesis. Reduced TAG lipase activity prevented the release of lipid molecules from lipid droplets.


As we planned to express PAH1 and DGA1 from the light-inducible promoter, we used another approach – a system of ordinary differential equations (i.e. an ODE model). This allowed us to follow the dependency of enzyme concentration on light intensity in a cell culture and to compare lipid accumulation in wild-type strain with our mutant strain where lipases were deleted and PAH1 and DGA1 were overexpressed.

Conclusions

● We improved TAG accumulation by inactivating TAG hydrolysis pathways.


● We further increased TAG content by light-inducible expression of human Plin3 gene.


● We functionalized the cells with light-harvesting nanoparticles and found that the biohybrids can use light to drive the lipid synthesis and therefore can maximise the carbon yield.

Human Practices

Advice regarding indium phosphide nanoparticles:

We were concerned about the safety of the indium phosphide nanoparticles that we used to coat the cell surface. Professor Junling Guo from Harvard University informed us that InP is one of the safest and most effective semiconductors for biohybrid engineering. Moreover, InP nanoparticles can be recycled for reuse.

Biohybrid assembly guidance:

Dr. Alexander Vanetsev from University of Tartu helped us to prepare the nanoparticle mixture for their following assembly on yeast cells.

Consultations on modeling:

Professor Junling Guo and Dr. Jens Hahn from Humboldt University of Berlin helped us to assess possible modifications in our modeling part.

Survey:
We conducted a public survey to estimate the potential customers’
1. demand for different lipid-containing products;
2. opinion on genetically modified organisms used in production;
3. opinion on the environmental footprint of the production.
The survey showed an increasing awareness of the younger generation about GMO products and the significance of environmental impact and also confirmed their demand for our lipid products.

Education , Public Engagement and Collaborations

iGEM is not only about the project but also about the community. This year is exceptional with all restrictions associated with COVID-19 pandemic. But nothing can stop iGEMers, even the biggest pandemic in the last 100 years. Throughout the year our team organized various workshops, scientific art exhibitions, escape room, designed comics, made an instagram rebuses, quiz, public survey, participated in many science fairs, and introduced the fundamental principles of synthetic biology to a wider audience of different ages and fields. We do not teach with complex over-the-head explanations, we teach while playing. During the season we have participated in 21 collaborations, 4 of which were initiated by us, and one leading to partnership. Even though most of them took place online, they still brought fun and many benefits.

Some of our Workshops:

Future Plans

SPARKLE provided proof of concept for a light-harvesting yeast biohybrid with indium phosphide nanoparticles. But to achieve greater yield and to improve our yeast biofactory, we would like to perform further modifications:

● Introduce overexpression cassette for PAP and DGAT enzymes, as their activity has been shown to increase TAG production.
● Substitute wild-type ACC1 with a constantly active mutant to increase the precursor supply.
● Replace ZWF1 deletion with a conditional shutoff to improve the cell’s growth rate.
● Develop a protocol to recycle the nanoparticles.
● Implement Our Team’s previous year project, where cell autolysis was achieved with the expression of bacterial glucanases.

Acknowledgements & Sponsors

Acknowledgements

Prof. Junling Guo
Dr. Jens Hahn
Dr. Alexander Vanetsev
Manana Modebadze

Sponsors

Acknowledgements & Sponsors

Acknowledgements

Prof. Junling Guo
Dr. Jens Hahn
Dr. Alexander Vanetsev
Manana Modebadze

Sponsors

Contact Us:

estoniatuit@gmail.com

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