Forbidden FRUITS

Cell Factory

Current industrial practices using fossil fuels add to the rising amounts of greenhouse gasses like CO2 in the atmosphere. Emission of greenhouse gasses is linked to climate change, resulting in rising sea levels and temperature changes (International Protocol on Climate Change, 2014)⁠. Therefore, more sustainable methods of producing chemicals have to be developed. Cellular systems are useful in the sustainable production of valuable chemical compounds, such as medicine or biofuels (Davy et al., 2017; Liao et al., 2016; Papagianni, 2012)⁠. Microorganisms can use renewable resources or even CO2 as feedstock. Taken together with the optimal energy use and minimal waste generation, the use of microorganisms as ‘cell factories’ is promising in making industrial practices more sustainable.

Strain instability

Cell factories often work well in the lab, but fare poorly in the real world. A limitation in the use of microorganisms to produce chemical compounds on large scale is the low efficiency and instability of genetically engineered microorganisms (Woodley et al., 2013). Life’s purpose of microbes is to grow as fast as possible, allocating as many resorts (e.g. nutrients and energy) as needed to do so. Cell factories, however, form products diverting energy to the production of a compound, which they do not need for growth. Therefore, they will eventually be outcompeted by faster-growing, but less productive, mutated microbes. This results in cell factories that are not scalable to industrial standards due to unstable production rates. While the production of compounds from these microbes can be environmentally friendly, and sometimes even CO2 negative, the problem of unstable production withholds companies from using them on a large scale. They then resort instead to the usage of environmentally unsustainable methods of producing these compounds (i.e. based on fossil fuels).

Our solution: Forbidden FRUITS

A solution to this problem is to couple product formation to microbial growth: by making the formation of the compound obligatory for growth, the production rate improves when the growth rate increases (Buerger, et al. 2019). Our scientific goal is to make a tool to design microbial production systems with stable, growth-coupled production. This tool not only allows for a fast development of stable microbial strains, which can produce valuable compounds, but also gives researchers the opportunity to explore the metabolic wiring of any microbe. A somehow more primitive version of this idea has been implemented before. It consisted on the usage of the ‘FRUITS’ (Finds Reactions Usable in Tapping Side products) algorithm, which identifies opportunities for growth coupled production within the metabolic network of a host organism (Du, et al. 2019). This algorithm is generic for any modelled microorganism but is limited to products which naturally occur in the host microbe (also called ‘native’ products). To enable the engineering of ‘tailor-made’ microbial production systems, we developed Forbidden FRUITS! The latter is not only generic for any microorganism, but also for any (non-native) product. It will predict the optimal combinations of genes to be knocked in and knocked out to yield stable production of any compound in any modelled microorganism: introducing ‘forbidden’ products in microbes with suitable traits. This conceptually means that we are expanding the selection of what we can produce, independent of which microbe is producing it. Salicylic acid (aspirin) made by E. Coli? We’re on it!


When the algorithm is given a desired product and the metabolic network of an organism, it computes several genetic engineering strategies, which should result in growth-coupled product formation. To verify the predictions of the algorithm, we will implement the genetic engineering strategy in different organisms: Synechocystis PCC6803(synechocystis), Escherichia coli (E.coli) and Synechococcus UTEX 2973 (UTEX 2973). We are aiming to produce salicylic acid in E.coli and synechocystis to show that Forbidden FRUITS can predict strategies for any microbial system. In addition, we will implement strategies to produce mannitol and lactate in synechocystis to show that Forbidden FRUITS can predict strategies for any product. For more information about the choices of the organisms and products, please visit our Proof of Concept page.

Our ‘pets’

The main focus of our research is on Synechocystis. Our motivation to use this microbe is connected to the fact that it is a phototrophic cyanobacteria, which is already used in synthetic biology (Vermaas, et al. 2001; Gale, et al. 2019). Because Synechocystis can directly convert atmospheric CO2 into products, it is a major step forward to a circular economy: as the CO2 emitted while using and transporting the product is used by the bacteria to form new products. Besides, we think we can learn from the ability of this microorganism to harvest energy from sunlight. We as a society are mining energy from all different resources, but cyanobacteria have been using sunlight for billions of years, so why not try to see if the industry can exploit this trait in making products for society? In addition to using Synechocystis, we use another cyanobacteria, Synechococcus UTEX 2973. This cyanobacteria has industrial relevance, for it has a higher growth rate than other cyanobacterial strains (Yu, et al. 2015).

Besides using phototrophs, we want to show that our algorithm can be applied in the organisms which are already used in different fields of biotechnology. Therefore, we used E.coli in our proof of concept. E.coli is one of the most commonly used microorganisms in biotechnology with a wide range of tools and applications (Idalia and Franco, 2017). By using E.coli we show that our algorithm can be readily applied to make the current biotech practices more sustainable and profitable.

Real world applications

With our project we want to make biotechnological processes more feasible and flexible. Our solution could have an enormous impact on the biotech sector and could provide a new tool in combating CO2 emissions. The production pathways of the products we have chosen to prove our concept illustrate different features of the algorithm. What these products have in common is the demand for more efficient and sustainable production processes. Our algorithm can offer a tool to overcome current limitations by being flexible in the production host and by offering a method to make any strain stably produce the compound. More information about possible applications and implementations of our products can be found on our implementation page.

Our products - background

We will introduce a production pathway for salicylic acid in Synechocystis and E.coli. The main reason for choosing salicylic acid as a target compound is because it requires a pyruvate dependent strain. The construction of such a strain allows for the rapid implementation of many other Forbidden FRUITS strategies for many other different products. This is because pyruvate can be seen as a central metabolic hub. And therefore, it is relatively easy to find reactions that link any given compound to it. This illustrates that a single Forbidden FRUITS strategy can be used to develop a chassis for the production of a variety of compounds. 

Salicylic acid is the benzoic precursor of the painkiller aspirin. Besides being used in the pharmaceutical industry, it is also applied in food preservation and cosmetics. Until present it is chemically synthesized using the Kolbe-Smith reaction (Brown, 2016). Naturally occurring in plants, an metabolic pathway to produce salicylic acid is readily available. Therefore, this compound has the potential of being produced by microbial systems.

Lactate serves as a resource for the production of bioplastics. Besides having great potential in the production of a biodegradable plastic, it can also be used in food, cosmetics and pharmaceuticals. Lactate can be produced by a chemical or fermentation process. Because of the increasing demand for lactate in its many applications, it is important to make the production process more efficient and sustainable (Abdel-Rahman, et al. 2011).

Mannitol is a sugar with application in the food, pharmaceutical and chemical industry. It can be produced from plant material or in a chemical process. Because of the environmental impact and the costs of both methods, the demand for a different production process is increasing (Madsen, et al. 2018). By implementing a mannitol production pathway, we show the possibility of producing this compound in a sustainable and cost effective manner. The mannitol production pathway is an example of a path optimized using the cheap lunch strategy. For more information about Cheap Lunch strategies, please visit our results page.


[1]Abdel-Rahman, M. A., Tashiro, Y., & Sonomoto, K. (2011). Lactic acid production from lignocellulose-derived sugars using lactic acid bacteria: Overview and limits. Journal of Biotechnology, 156(4), 286–301.

[2]Buerger, J., Gronenberg, L. S., Genee, H. J., & Sommer, M. O. A. (2019). Wiring cell growth to product formation. Current Opinion in Biotechnology, 59, 85–92.

[3]Brown, W. H. (2016). Salicylic Acid. Encyclopaedia Britannica. Accessed Jun 15, 2020.

[4]Davy, A. M., Kildegaard, H. F., & Andersen, M. R. (2017). Cell Factory Engineering. Cell Systems, 4(3), 262–275.

[5]Du, W., Jongbloets, J. A., Van Boxtel, C., Pineda Hernández, H., Lips, D., Oliver, B. G., Hellingwerf, K. J., & Branco Dos Santos, F. (2018). Alignment of microbial fitness with engineered product formation: Obligatory coupling between acetate production and photoautotrophic growth. Biotechnology for Biofuels, 11(1), 1–13.

[6]Gale, G. A. R., Osorio, A. A. S., Mills, L. A., Wang, B., Lea-Smith, D. J., & McCormick, A. J. (2019). Emerging species and genome editing tools: Future prospects in cyanobacterial synthetic biology. Microorganisms, 7(10).

[7]Idalia, V.-M., & Franco, B. (2017). Escherichia coli as a Model Organism and Its Application in Biotechnology. In A. Samie (Ed.), Escherichia Coli: Recent advances on Physiology, Pathogenesis and Biotechnological applications (pp. 253–274).

[8]International Protocol on Climate Change. (2014). Climate Change 2014 Synthesis Report: Summary Chapter for Policymakers. Ipcc, 31.

[9]Liao, J. C., Mi, L., Pontrelli, S., & Luo, S. (2016). Fuelling the future: Microbial engineering for the production of sustainable biofuels. Nature Reviews Microbiology, 14(5), 288–304.

[10]Madsen, M. A., Semerdzhiev, S., Amtmann, A., & Tonon, T. (2018). Engineering Mannitol Biosynthesis in Escherichia coli and Synechococcus sp. PCC 7002 Using a Green Algal Fusion Protein. ACS Synthetic Biology, 7(12), 2833–2840.

[11]Papagianni, M. (2012). Metabolic engineering of lactic acid bacteria for the production of industrially important compounds. Computational and Structural Biotechnology Journal, 3(4), 1–8.

[12]Riekhof, M.-C., Regnier, E., & Quaas, M. F. (2019). Economic growth, international trade, and the depletion or conservation of renewable natural resources. Journal of Environmental Economics and Management, 97, 116–133.

[13]Song, A. A. L., In, L. L. A., Lim, S. H. E., & Rahim, R. A. (2017). A review on Lactococcus lactis: From food to factory. Microbial Cell Factories, 16(1), 1–15.

[14]Vermaas, W. F. J. (2001). Photosynthesis and Respiration in Cyanobacteria. In eLS.

[15]Woodley, J. M., Breuer, M., & Mink, D. (2013). A future perspective on the role of industrial biotechnology for chemicals production. Chemical Engineering Research and Design, 91(10), 2029–2036.

[16]Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., Koppenaal, D. W., Brand, J. J., & Pakrasi, H. B. (2015). Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO₂. Scientific Reports, 5, 8132.

Forbidden FRUITS

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