Description
We are developing a circular economy project based on the biosynthesis of resveratrol in Escherichia coli, using precursors present in by-products from the wine industry. Resveratrol is a natural molecule found in wine and grapes with important antioxidant effects, as well as protective properties against certain cancers. In plants, it is part of an immune system that is triggered when there is an infection from a pathogen.
Therefore, our main challenge is to accomplish the efficient bioproduction of this important molecule, based on values such as respect for the environment, local production and the future improvement of human health and nutrition. In order to achieve this goal, we will try to develop the natural biosynthesis pathway in our bacteria.[1][2]
The project selection
Life expectancy has seen a significant increase, thanks to advancements in the fields of medicine, nutrition, biology, and pharmacy. We've achieved adding a few more years to our lives (which is impressive) but now the challenge is to guarantee that those years are high quality years.
We are concerned that our society is dealing with the consequences of overpopulation and its increasing longevity. According to the statistical office of the European Union (Eurostat), in 2022, more than one fifth (21.1 %) of the EU population was aged 65 and over. This demographic change is directly responsible for the EUâs age pyramid inversion, with a noticeable increase of the older population. In addition, this growing population is related to the development of unhealthy lifestyles with poor nutrition, which can cause health problems including obesity or chronic diseases.
Because of that it's crucial to understand there are illnesses directly associated with aging and there's a necessity of dealing with those issues to improve the elderly's population standard of living. Some examples of health problems that are characteristic of that elderly population are cardiovascular diseases, neurodegenerative illnesses (such as Parkinson's disease or Alzheimer's disease) , cancer, diabetes and chronic inflammation among others.
The benefits of resveratrol in human health transform this bioactive compound into a powerful tool to overcome the pernicious effects of aging. Plenty of studies are nowadays supporting the benefits of consuming resveratrol (even though it's still on constant research) due to its capacity to lower LDL cholesterol, its neuroprotective properties, the fact that it can help to inhibit the growth of cancer cells and also that it seem to be beneficial in the treatment of patients with diabetes (owing to its ability to improve insulin sensitivity, regulate glucose blood levels and protect pancreatic cells).
Furthermore, the impact of the winery industry waste demands the introduction of reuse procedures, such as resveratrol reuse. Lastly, the great amount of findings and evidence about resveratrol-related health benefits makes it the right time to tackle this challenge. [3]
Besides, we were also inspired by the French paradox. This describes the phenomena of how in France, even though they consumed high amounts of fatty acids and had high cholesterol diets, they did not suffer from the same amount of cardiovascular issues as other regions with similar food intake options. This fact surprised the scientific community and was explained by the daily moderate consumption of red wine, which contains resveratrol and other powerful antioxidants. This is how resveratrol was discovered and we found extremely fascinating how it seems to improve health in that area.
Moreover, in our autonomous community, Navarra, the wine sector is one of the most important. The numbers from the Market Information System for the Wine Sector of the Ministry of Agriculture of Spain estimate production at 83.18 million liters, which includes wines with a certificate of origin, in addition to those marketed under any other modality, as packaged or in bulk. Specifically, based on Navarre production, we have up to six protected wines with a certificate of origin in our territory. [4]
Additionally, according to data from the International Organisation of Vine and Wine (OIV), it is estimated that 100 kg of grapes generate about 25 kg of waste, 50% being the skin of the grape, and the rest stems and seeds.
Chemical extraction vs. new method of obtaining resveratrol
The amount of resveratrol obtained through chemical extraction from red grapes and their byproducts varies widely, influenced by factors like extraction methods and processing conditions. It is expressed as a percentage relative to the resveratrol content in grape skins, ranging from 20% to over 90%. The yield depends on the extraction efficiency, grape variety, ripeness, and other factors, with precise results contingent on specific protocols used in each case.
Grape pomace (GP), the primary residue of winemaking, contains skins and seeds remaining after fermentation and maceration. This byproduct has substantial polyphenol levels due to incomplete extraction during winemakin, making it an alternative source of natural antioxidants. GP utilization aligns with industry efforts to reduce environmental impact through byproduct valorization. Consequently, significant attention has focused on recovering bioactive phenolics from grape by-products for application in pharmaceutical and food industries. [5]
For the valorization of viticultural products, another researched tool is bioproduction using microorganisms, particularly E. coli. Significant variability has been observed in the bioproductions obtained in different studies. Nevertheless, the highest production with our genes (referring to 4CL1 and STS from the organisms we have taken) is 2340 mg/L. [6]
In addition, the maximum bioproduction, apart from being achieved through the use of our genes, is attainable by introducing p-coumaric acid (chemically in their case). However, our objective is to achieve this utilizing by-products.
Inspirations
Our project has been inspired by other iGEM teams that have worked with resveratrol in the past, including Rice_University 2008, UPPsala 2013, Tel-Hai 2017 and Virginia 2020.
Previous teams raised resveratrol biosynthesis as a possible application for their project, such as the production of probiotics, beer or wine. For us, the main focus is resveratrol and its possible benefits for human health and nutrition. In addition, we aim to use the circular economy for this, using waste from the wine industry as a substrate to develop the project.
Rice University 2008
The Rice University team's project (2008) was the biosynthesis of resveratrol in microorganisms responsible for the alcoholic fermentation of beer (biobeer), with the aim of obtaining resveratrol-rich beer, and to take advantage of the massive consumption and commercial pull that this has in the world, in a very intelligent way. Like our team, they are committed to resveratrol because of its beneficial health properties, and they share our interest in the biosynthesis of the compound using synthetic biology. They therefore used yeast, and added only two optimised genes: TAL and 4CL (our three: STS, TAL and 4CL). In addition, they considered the quantitative evaluation of the final product by HPLC and came up with a very interesting construct design.
This team inspired us in the selection of the project given the great potential of resveratrol, although we wanted to distinguish ourselves by focusing on the compound itself and on the circular economy.
UPPSala 2013
The Uppsala team's project (2013) aimed to modify the bacterium Lactobacillus to transform it into a probiotic platform for metabolic engineering. This involved altering Lactobacillus to design probiotics capable of producing beta-carotene, resveratrol, miraculin, and saffron. The current project our team is undertaking this year shares similarities with the 2013 project but with a key difference: we are focusing exclusively on resveratrol synthesis and have chosen to use the bacterium Escherichia coli as the host organism instead of Lactobacillus.
The selection of Escherichia coli is based on several inherent advantages of this bacterium. Firstly, its ease of cultivation in the laboratory simplifies large-scale production. Additionally, its rapid growth can result in doubling times as short as half an hour under optimal conditions, enabling the attainment of high cell densities in a relatively short period.Another significant advantage of Escherichia coli is its cost-effectiveness in biomolecule production due to its ease of cultivation and rapid growth. Furthermore, its genetic stability ensures that genetic modifications introduced into the bacterium remain intact throughout cell divisions.
In summary, the choice of Escherichia coli as the host organism is due to its suitability in terms of ease of cultivation, rapid growth, and genetic stability, making it an attractive option for resveratrol production in our current project.
Tel-Hai 2017
The TeI-Hai team's project (2017) focuses on wine improvement by using synthetic biology. To do so, they decided to add new characteristics to the yeast S. cerevisiae, the main microorganism involved in the wine fermentation process.
The issues they tried to solve were the frequent bacterial and fungal infections and the high alcohol concentrations of wine products. On one hand, they tried to minimize the presence of the yeast Brettanomyces bruxellensis (Brett), which is one of the yeast responsible for the deterioration of wine during ageing. To do this, they set out to insert a gene for a Brett-specific toxin into the genome of their S. cerevisiae yeast. They also aimed to direct the biochemical pathway of alcohol production towards the glycerol production pathway, in order to reduce the level of alcohol. In addition, they decided to improve other properties of the wine, such as a lower glycaemic index and improved nutritional values for the benefit of the consumer's health. For this reason, they tried to introduce the Miraculin molecule, which has a zero glycaemic index and is acceptable for diabetics, as well as making the yeast produce resveratrol.
This project shares a common interest in synthesizing resveratrol with the help of synthetic biology. However, TeI-Hai's team set out to express resveratrol, along with other molecules, in yeast (S. cerevisiae) rather than in bacteria (E.coli in our case). Like us, they used the TAL, 4CL and STS genes involved in the metabolic pathway of resveratrol, but optimized them for yeast. Expressing multiple molecules in yeast led them to the construction of multiple parts, with plasmid vectors, promoters, terminators and other components different from those used by our team.
Virginia 2020
The Virginia team project idea was to create a platform which could improve the efficiency of biosynthesis in a modified organism, the main idea was optimizing the metabolic flux. This idea was specially created for E. coli with the ability to synthesize resveratrol. We loved the idea of working with resveratrol and E coli but we wanted to bring it closer, link it up with a local problem.
References
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| [4] |
Viticultura y enologĂa. Zonas vitivinĂcolas - navarra.es. (s. f.). Retrieved June 2023 |
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| [6] |