In SPARKLE we decided to put our integrated human practices into the 3R strategy framework that would later help us carry out our project in a way that would best serve the society. 3R strategy provides a set of questions, each of which falls under one of the three categories: Reflection, Responsibility, and Responsiveness ("Doing Human Practices During a Global Pandemic", 2020).
At the beginning of the project, we answered all the questions explicitly and committed to updating the answers as we moved along. Working based on the 3R strategy helped to continue the integrated human practices in a structured manner, making it easier to plan our work and understand our project’s impact on the world and the environment.
Because in SPARKLE we were designing a platform for high-output lipid production instead of a product with a single immediate application, we thoroughly researched relevant industries and assessed the potential impact that our product could have on them.
Advice: Mrs. Modebadze confirmed that our strategy is eligible and provided us with a more complete list of lipid products. She advised us to get even more information regarding the product necessity from pharmaceutical companies potentially interested in our project idea.
Our implementation: Once we collected the expert’s opinion, we decided to validate it by conducting a public survey. We included the resulting list of lipid-based products in the public survey.
Environmental impact of the product is important for respondents
People are familiar with GMO concept
According to the results, some of the lipid derived products are more widely used than others.
Since we were engineering a biohybrid system for large-scale production of lipids, we were concerned about the use of indium phosphide as a semiconductor, as we found that it could pose potential health risks as well as have some negative impact on the environment.
Advice 1: "I reckon InP is the ideal semiconductor material for your application due to the visible light-matching band gap and biocompatibility. Besides, we only used the InP as nanoscale form for the engineering of biohyrbids, so the total future usage of this material should be still far below the amount in nature resourced.”
Our implementation: We decided to proceed with the use of InP as a photosensitizer. As we learned about the possibility to recycle used InP nanoparticles, we proved our project to be eco-friendly.
Advice 2: Prof. Guo provided us with a calculation method that can be used to estimate NADPH/NADP+ ratio in the cell, which in principle could be used as a proxy for the electron supply by photosensitized InP nanoparticles. However, according to Junling, the electron transfers from semiconductor to the cytosol, and eventually the enzyme is a complex process which is not yet fully understood.
Future implementation: After further research, we discovered that although these aspects will certainly affect the final lipid yield, they are difficult to model mathematically using the available tools. Nevertheless, modelling the effect of light on lipid production in the strain remains in our plans, as it may predict optimal conditions that will maximize production efficiency.
Advice 1:"Grinding InP particles in solution to induce the Rehbinder effect.
Our implementation: We took his advice and ground the InP in water solution to make the process easier.
Advice 2: Start with higher initial InP suspension volume and repeat centrifugation several times at two different speeds.
Our implementation 2: We repeated our centrifugation for several times at either 8000xg for 5 min (supernatant is collected) and 10000x g for 15 min (pellet is collected) to maximize nanoparticles yield. We also started with a bigger volume of InP suspension to make the separation of particles of different sizes more efficient. In the original article, researchers used 1.5 ml tubes, while we performed initial centrifugations in a 50 ml falcon tube.
Advice: The deletion of the TAG lipase reaction in the agent-based model ‘would affect the system more than the mere knockout of the TGLx’.
Our implementation: The comments from Dr. Jens Hahn helped us to correctly address our assumptions about the role of TAG lipase in the model.
Important 3R findings:
Our yeast strain provides an alternative to plant-, animal- and chemically derived lipids.
Many different industries, such as the pharmaceutical, cosmetic, and food industry, could benefit from our project.
Some environmental concerns needed to be addressed regarding the safety and sustainability of indium phosphide.
Impact:
Relevant industry: We continued searching for further applications for TAGs produced by our strain. We found that due to its genetic tractability and a large amount of accessible data, S. cerevisiae is an ideal model organism for the expression of heterologous genes involved in the biosynthesis of unsaturated and polyunsaturated fatty acids (PUFAs). This makes S. cerevisiae a useful platform for the biotechnological production of PUFAs. Recently, many genes involved in the PUFA biosynthesis have been identified and sequenced (Uemura, 2012). The following genes have been successfully introduced into S. cerevisiae to produce unsaturated and polyunsaturated fatty acids.
Genes
Fatty acids
Potential applications
References
Desaturase genes FAD2, FAD3, KlFAD2, lFAD2
Linoleic, α-linolenic, and γ-linolenic acids
There is evidence that PUFAs can alleviate the symptoms of many chronic diseases (Uemura, 2012)
Ratledge & Wilkinson (1988), Kainou et al. (2006)
Desaturases + elongases+cytochrome b5
long-chain PUFAs arachidonic acid, arachidonic acid, eicosapentaenoic acid (EPA), eicosapentaenoic acid (DHA)1
Long-chain PUFAs are used as a food supplement for normal development and maintenance of human health (Uemura, 2012)
Vasconcelos et al. (2019), Beaudoin et al. (2000), Meyer et al. (2004), Pereira et al. (2004)
Δ12-hydroxylase (FAH12)
Rsicinoleic acid
Can be used as a potential petrochemical replacement in a variety of industrial processes (Uemura, 2012)
Smith et al. (2003), Meesapyodsuk & Qiu (2008)
Therefore, we envision that after additional genetic modifications our final yeast strain will serve as a platform to produce useful fatty acids such as ⍵-3 and ⍵-6.
List:
Soap
Shampoo
Shower gel
Hair oil
Body oil
Makeup removal
Face cream
Lipstick
Foundation
Motor oil
Lubricant
Candle
Omega 3
Omega 6
Omega 9
Vitamin A
Suppository
While we were researching the topic, we found that: although InP appears to be safer than Cd-based semiconductor materials, several independent organizations (IARC, REACH, AGS, and BMAS among them) have concluded that InP is a hazardous substance
InP was listed on the 2017 list of Critical Raw Materials for the EU because of the potential risks of a supply shortage.
Taking this into account, we reasoned that using InP in industrial manufactory would lead to increasing demand for the raw material which may eventually lead to exhaustion of its natural reserves. We decided to ask for advice on this topic from Prof. Guo, an author of the paper Light-driven fine chemical production in yeast biohybrids.
One of the findings from the 3R strategy was that the InP nanoparticles may pose a risk on health as well as on the environment. With this question we reached out to Prof. Junling Guo, the author of the paper Light-driven fine chemical production in yeast biohybrids (Guo, J. et al, 2016).
Prof. Guo has informed us that InP is one of the safest semiconductors for biohybrid engineering. It is one of the few materials that would have similar electronic properties and good biocompatibility. Prof. Guo assumed that InP can be recycled and reused in different fermentation batches which makes it one of the most sustainable and green semiconductors suitable for similar applications.
According to Prof. Junling Guo, a different approach is to utilize electrodes as an alternative electron source. This has indeed been a well-established methodology in the field of biohybrid engineering, which is supported by a variety of available literature on this topic.
One of the parts that we decided to focus on this year is modelling our optogenetic system and lipid production under our experimental conditions. Light in our system represents a major input signal, and its effect is two-fold. Firstly, it influences the expression of certain genes in our overexpression plasmid. Secondly, it plays a key role as an energy source for the cell (photosensitized InP nanoparticles generate electrons). While the former part could be modelled with a set of ODEs, the latter one seemed less straightforward. Since we were interested in how exactly light properties would affect the electron generation by the semiconductor and subsequent NADP+ regeneration, we again asked Prof. Guo for help.
Rehbinder effect is induced by absorption and results in a reduction of metal strength. The absorption-active components participate in interatomic bond rupture, making the grinding process easier (A. I. Malkin, 2011).
Dr. Vanetsev suggested using a bigger initial InP suspension volume to increase efficiency. Regarding the separation of InP nanoparticles by size, he said that although ~90% of the bigger nanoparticles stay in the pellet and small nanoparticles should stay on the walls, there are about 50% small nanoparticles in the pellet as well. Therefore, he advised us to repeat the centrifugation several times at different speeds to have most of the smallest nanoparticles left on the walls.
Presumably, we can compare this effect to the knockdown of the lipases in the wet- lab and use the agent-based model. Dr. Jens Hahn, the coauthor of the article Computational Modelling of Lipid Metabolism in Yeast (Schützhold et al., 2016), specified that TGL4 was chosen for the agent-based model, as it “has several functions in lipid metabolism (see also Rajakumari & Daum, 2010)” and its knockdown possibly could be a threshold for the effective lipid accumulation. Dr. Jens Hahn also directed us to the recommended following research on genome scale metabolic model, which could better answer our questions regarding TAG lipases activity in the S. cerevisiae. One of the approaches used in our modelling part was an agent-based model. We were interested in the comparison of our model with the actual results we got in the lab. The knockdown of both TGL1 and TGL4 lipases in the agent-based approach and actual deletion of TGL 3, TGL 4, and TGL 5 in the wet-lab drastically affect the system and rapidly increase the accumulation of lipids in the lipid droplets. Dr. Jens Hahn’s reply clarified the functions and possible limitations of using the model for our project.
Tao, Bernie Y. (2007). Industrial Applications for Plant Oils and Lipids. Bioprocessing for Value-Added Producs from Renewable Resources, Chapter 24, 611-627. https://doi.org/10.1016/B978- 044452114-9/50025-6 Guo, J. , Tardy, B., Christofferson, A. et al. (2016). Modular assembly of superstructures from polyphenol-functionalized building blocks. Nature Nanotechn, 11, 1105-1111. https://doi.org/10.1038/nnano.2016.172 Malkin, A. I. (2011). Regularities and Mechanisms of the Rehbinder’s Effect. Kolloidnyi Zhurnal,74(2), 223-238. https://doi.org/10.1134/S1061933X12020068 Beaudoin, F., Michaelson, L. V, Hey, S. J., Lewis, M. J., Shewry, P. R., Sayanova, O., & Napier, J. A. (2000). Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6421–6426. https://doi.org/10.1073/pnas.110140197 Kainou, K., Kamisaka, Y., Kimura, K., & Uemura, H. (2006). Isolation of Δ12 and ω3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and α-linolenic acids in Saccharomyces cerevisiae. Yeast, 23(8), 605–612. https://doi.org/10.1002/yea.1378 Meesapyodsuk, D., & Qiu, X. (2008). An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis. Plant Physiology, 147(3), 1325–1333. https://doi.org/10.1104/pp.108.117168 Meyer, A., Kirsch, H., Domergue, F., Abbadi, A., Sperling, P., Bauer, J., Cirpus, P., Zank, T. K., Moreau, H., Roscoe, T. J., Zähringer, U., & Heinz, E. (2004). Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis. Journal of Lipid Research, 45(10), 1899–1909. https://doi.org/10.1194/jlr.M400181-JLR200 Pereira, S. L., Leonard, A. E., Huang, Y.-S., Chuang, L.-T., & Mukerji, P. (2004). Identification of two novel microalgal enzymes involved in the conversion of the omega3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid. The Biochemical Journal, 384(Pt 2), 357–366. https://doi.org/10.1042/BJ20040970 Ratledge, C., & Wilkinson, S. G. (1988). Microbial lipids (Vol. 2). Academic Pr. Smith, M. A., Moon, H., Chowrira, G., & Kunst, L. (2003). Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta, 217(3), 507–516. https://doi.org/10.1007/s00425-003-1015-6 Uemura, H. (2012). Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives. Applied Microbiology and Biotechnology, 95(1), 1–12. https://doi.org/10.1007/s00253-012-4105-1 Vasconcelos, B., Teixeira, J. C., Dragone, G., & 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. https://doi.org/10.1007/s00253-019-09742-x Rajakumari, S., & Daum, G. (2010). Multiple functions as lipase, steryl ester hydrolase, phospholipase, and acyltransferase of Tgl4p from the yeast Saccharomyces cerevisiae. Journal of Biological Chemistry, 285(21), 15769–15776. https://doi.org/10.1074/jbc.M109.076331 Schützhold, V., Hahn, J., Tummler, K., & Klipp, E. (2016). Computational modeling of lipid metabolism in yeast. Frontiers in Molecular Biosciences, 3(SEP). https://doi.org/10.3389/fmolb.2016.00057 Doing Human Practices During a Global Pandemic. (2020). Retrieved 24 October 2020, from https://www.youtube.com/watch?v=w79IygjwXEM&feature=youtu.be
