Team:Duesseldorf/Description

    • Our Inspiration

    As early as from the very first meeting to form the current iGEM Team Düsseldorf, the urge was expressed to improve the environment and reform an everyday product that is used by a majority of humans around planet earth, but hardly anyone is aware of its environmental impact. Therefore two weeks of intense foundational research of every-day products were conducted, to elucidate the amount of usage and their impact to the environment and the society. After two weeks of research, the team came up with several ideas, but there was only one consensus: A major group of people consumes dairy products and loves the taste of it.

    When drinking a cold, fresh glass of milk, nobody thinks of all the damage and negative consequences that the consumption of this product entails. More and more people are becoming aware of the causes of dairy farming and greenhouse gas emissions, but the vast majority does not know that cows are involved to a large extent. However, it cannot be the solution to stop using milk and all other dairy products completely, since it is firmly rooted in our society and culture. It is therefore unavoidable to find a solution that does not only reduce emissions, but which is also a safe and healthy product with all the benefits of traditional cow’s milk (including taste), but without drawbacks such as antibiotics and other additives. The idea of an environmentally friendly, sustainable and ethically acceptable milk alternative was born.

    All proteins and other nutrient sources contained in traditional milk are found in our SynMylk in their purest form. Why? In contrast to plant alternatives, our aim is to create a product that is identical to traditional cow’s milk. In SynMylk, no traces of antibiotics or other contaminants will be detectable and it is also lactose-free by default.

    Our Project Description

    As part of the competition “international Genetically Engineered Machine” (iGEM), the team at the Heinrich Heine University in Düsseldorf has set itself the task of producing cow's milk without animal products. The dairy industry has a major impact on the environment, as the cows release methane during digestion which is 25 times more harmful than carbon dioxide1, in addition to an enormous carbon footprint due to feed production, grazing lands that could otherwise be used for food crops, and water usage. We are trying to create an alternative to cow’s milk with a product that has the same taste, consistency and nutritional value. Current plant-based milk alternatives such as soy or almond milk are not currently achieving the goal of replacing cow's milk, as they differ too much in taste and consistency. Our goal will be achieved by heterologous expression of proteins, enzymes, metabolic engineering, mathematical modeling and many other methods of synthetic biology. In all these areas we are supported by various institutes in Düsseldorf and Jülich with expertise to make our project as successful as possible.

    Fig. 1: Main components in cow´s milk.
    Cow's milk consists of water, lactose, lipids, proteins, macro- and microminerals, all of which are emulsified in water. Nutrients can be purchased as pure chemical syntheses, but the proteins and lipids from milk are not commercially available without using milk isolates. These two components are our main focus.

    Milk proteins are divided into two fractions; casein and whey proteins. Caseins are made up of four proteins (ɑ-s1-, ɑ-s2-, β- and κ-casein). Whey proteins contain hundreds of different proteins, so we will focus on the most abundant one, ɑ-lactalbumin. After proof-of-concept production in Escherichia coli we will use Bacillus subtilis as our main production organism to secrete the proteins into its culture medium2 with the help of diverse signal sequences. Additionally, we plan to explore the cyanobacterium Synechocystis sp. PCC 6803 as a new promising host for protein production in a photosynthetic organism, yielding a net-reduction of CO23.

    Fatty acids, which are the chain-length defining components of lipids are synthesized in the cell in an iterative process, extending a carbohydrate chain by two carbon atoms each time. A thioesterase enzyme terminates this process at a specific length, yielding a free fatty acid to be used in a triglyceride lipid4. To produce the full range of lipids in milk, we first need to synthesize the short-chain (C4:0 to C13:0) fatty acids by heterologous expression of different thioesterases5. While many longer chain fatty acids are naturally produced by most known model microorganisms, our long term goal is to increase the yield of these as well. We plan to achieve this goal by engineering multiple different production strains.

    Fig. 2: Fatty-acid metabolism of Synechocystis sp. PCC 6803.

    We chose four thioesterases from several organisms, including microalgae, plants6 and bacteria5, 7, which have previously been reported to determine different chain lengths for fatty acids. Because of this, they will be heterologously expressed in Synechocystis sp. PCC 68038. To improve the overall synthesis of all fatty acids of interest, we will overexpress the enzymes needed for the ACCase complex. This complex is the rate-limiting factor of the fatty acid synthesis9, 10, since it catalyzes the production of the precursor for all fatty acids, malonyl-CoA. It is planned to do this in E. coli and will be tested in it to analyze the yield and efficiency - however, our long term goal is to eventually implement all our design strategies in a cyanobacterial host. Further improvement will be done by a CRISPRi/dCas9 - system which is already established for Synechocystis sp. PCC 68038. We will down-regulate enzymes involving beta-oxidation, elongation-steps or leading towards other off-target pathways11, 7. Finally, by cloning different biosensors with different promoters, like PfadBA11, PaldA, or the FadR-dependent reporter system11, which are induced by different acyl-CoA or free fatty acids, we want to create a simple method to quantify fatty acid synthesis. Sensing these molecules, results in the expression of selektorgens as chromoproteins or the fluorescent protein eYFP and the fatty acids can be read out in a plate reader.

    References
    1. https://www.umweltbundesamt.de/
    2. Fu, Ling & Xu, Zi & Li, Weifen & Shuai, Jiang & Lu, Ping & Hu, Chun. (2007). Protein secretion pathways in Bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnology advances. 25. 1-12. 10.1016/j.biotechadv.2006.08.002.
    3. Quinn, Jason C., and Ryan Davis. "The potentials and challenges of algae based biofuels: a review of the techno-economic, life cycle, and resource assessment modeling." Bioresource technology 184 (2015): 444-452.
    4. PFLEGER, Brian F.; GOSSING, Michael; NIELSEN, Jens. Metabolic engineering strategies for microbial synthesis of oleochemicals. Metabolic engineering, 2015, 29.
    5. Jawed, Kamran & Mattam, Anu & Fatma, Zia & Wajid, Saima & Abdin, M. & Yazdani, Syed Shams. (2016). Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway. PLoS ONE. 11. 10.1371/journal.pone.0160035.
    6. Katayoon Dehesh, Aubrey Jones, Deborah S. Knutzon, Toni A. Voelker Production of high levels of 8: 0 and 10: 0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana. The Plant Journal, 1996, 9. Jg., Nr. 2, S. 167-172.
    7. Xiping Liu, Haiying Yu, Xu Jiang, Guomin Ai, Bo Yu, Kun Zhu Biosynthesis of butenoic acid through fatty acid biosynthesis pathway in Escherichia coli. Applied microbiology and biotechnology, 2015, 99. Jg., Nr. 4, S. 1795-1804.
    8. LIU, Xinyao; SHENG, Jie; CURTISS III, Roy. Fatty acid production in genetically modified cyanobacteria. Proceedings of the National Academy of Sciences, 2011, 108. Jg., Nr. 17, S. 6899-6904.
    9. Cao, Yujin & Cheng, Tao & Zhao, Guang & Niu, Wei & Guo, Jiantao & Xian, Mo & Liu, Huizhou. (2016). Metabolic engineering of Escherichia coli for the production of hydroxy fatty acids from glucose. BMC Biotechnology. 16. 10.1186/s12896-016-0257-x.
    10. Chandran Sathesh-Prabu, Kwang Soo Shin, Geun Hwa Kwak, Sang-Kyu Jung, and Sung Kuk Lee Microbial Production of Fatty Acid via Metabolic Engineering and Synthetic Biology. Biotechnology and bioprocess engineering, 2019, 24. Jg., Nr. 1, S. 23-40.
    11. Lun Yao, Ivana Cengic, Josefine Anfelt and Elton P. Hudson Multiple gene repression in cyanobacteria using CRISPRi. ACS synthetic biology, 2015, 5. Jg., Nr. 3, S. 207-212.