Team:AISSU Union/Description
Overview：
In the contemporary society, the issue of population aging is becoming increasingly and having cognitive problems has posted serious threats to the seniors’ health.
Our team members are trying to mitigate this problem by applying Saccharomyces cerevisiae to produce a type of flavanol, which is a chemical that has proved to have significant effects on seniors’ cognitive abilities. We also understand that there are limited ways to produce our final product—catechin. There are some techniques that have been proposed to extract flavanols in plants, such as maceration, percolation, hydro-distillation, boiling, reflux, soaking, and soxhlet (Alara et al., 2018a). However, these techniques are characterized by the use of large amounts of highly-purified organic solvents, lower extraction yields, low selectivity, long extraction times, thermal degradation of target compounds, associated environmental concerns and costs, compared to other techniques. In addition, For most of those articles that describe how to apply microorganisms to produce flavanols, it was E.coli that was used in the process. (Zhao, S., et al) As a result, we choose Saccharomyces cerevisiae to convert L-tyrosine (Tyr) to catechin to produce a kind of “memory bread” as our project to achieve our ultimate goal, which is to produce catechins and fermentate our bread simultaneously with only one type of Saccharomyces cerevisiae, so that we can produce our'' memory bread'' without going through E. coli. Therefore, we choose Saccharomyces cerevisiae to convert L-tyrosine (Tyr) to catechin to produce a kind of “memory bread” as our project.
Inspiration:
The reason we want to do this project is that aging population is becoming a serious issue in many countries like Japan, the USA and China. Take China as an example, the aging of Chinese population is the result of the continuing decline in fertility rates and increased life expectancy. To be specific, the anti-natalist policy implemented by the Chinese government (the one-child policy) has caused a gradual decrease in birth rate of China, resulting in higher proportion of old people.(Fig1.)
Fig1: The percentage of old people in China from 2013 to 2018(http://5b0988e595225.cdn.sohucs.com/images/20191103/52169ebb659c44bfa0783bcf938ff5a8.png)
From some case studies and research, we have learned some key facts of aging population around the world and they are following below:
Between 2015 and 2050, the proportion of the world's population over 60 was predicted to nearly double from 12% to 22%.
By 2020, the number of people aged 60 years and older will outnumber children younger than 5.
In 2050, 80% of older people will be living in LEDCs and NICs.
The pace of population ageing is much faster than in the past.
(https://www.who.int/news-room/fact-sheets/detail/ageing-and-health)
The aging population can lead to different social problems. First and foremost, it puts pressure on the social welfare system. According to statistics, by the end of 2008, China had 37,623 elderly welfare institutions and 2.45 million old-age beds, accounting for only 1.5% of the elderly population over 60 of age, which is not only lower than the figure (5%-7%) in developed countries, but also lower than the figure (2%-3%) in some developing countries.
(https://baike.baidu.com/item/%E4%BA%BA%E5%8F%A3%E8%80%81%E9%BE%84%E5%8C%96/1980305)
Furthermore, the risk for the old people undergo diseases and disorders like subjective cognitive decline increases with age.
Subjective Cognitive Decline (SCD) is the self-reported experience of worsening or more frequent confusion or memory loss. SCD can have implications for living with and managing chronic disease, or performing everyday activities like cooking or cleaning. (Fig2.)
Fig2: An old man suffering from subjective cognitive decline(SCD)
Cognition is a combination of processes in the brain that includes the ability to learn, to remember, and to make judgments. When cognition is impaired, it can have a profound impact on an individual’s overall health and well-being. Cognitive decline can range from mild cognitive impairment to dementia, which is a form of decline in abilities that is severe enough to interfere with daily life. Relevant studies have shown that:
The prevalence of subjective cognitive decline (SCD) is 11.1%..
The prevalence of SCD among adults aged 65 and older is 11.7% compared to 10.8% among adults aged 45-64.
The prevalence of SCD is 11.3% among men compared to 10.6% among women.
The prevalence of SCD differs among racial/ethnic groups, 10.9% of Whites report SCD compared to 12.8% of African Americans, 11.0% of Hispanics, and 6.7% of Asians and Pacific Islanders.
Lower prevalence of SCD is reported among adults with more years of formal education.
( https://www.cdc.gov/aging/data/subjective-cognitive-decline-brief.html)
We can take the USA as an example. (Fig3.)
Fig3: The percentage of adults with SCD, who discussed their symptoms of memory loss and confusion with a health care professional in the USA
Those studies reflect the fact that there are a relative high proportion of the older generations suffering from SCD and this phenomenon is becoming more and more severe. This further makes us recognize the seriousness of the problem. This is exactly the original intention of our project.
Also, our team members have learned that old people do not like to eat healthcare products because they believe that they would do more harm than good. By contrast, they like to eat food made from flour and wheat, especially for the seniors who are living in the northern part of China. Most of the old people like to have bread (Fig4.) and porridge for breakfasts. The reason they eat bread and porridge is pretty simple: according to their consensus to the food , bread and porridge are macrobiotic food so they are in favor of them. Because yeast is not involved in the production of porridge, we choose bread as a vector to load catechin to ensure that the elderly have a more comfortable intake of catechin.
Fig4: Bread
Substrate and products:
We use L-tyrosine (Tyr) as our substrate to produce naringenin as an intermediate product and catechin as our final product. All products and central molecules we produced are members of flavonoids.(Fig5.)
Flavonoids are among the phytochemicals with proven activity towards the prevention of aging-related diseases, including the treatment of nervous and cardiovascular diseases, Parkinson’s and Alzheimer’s disease etc. Chemically, all flavonoids possess general structure of a 15-carbon skeleton with two phenyl rings (rings A and B) connected by a heterocyclic ring (ring C). In flavonoids biosynthetic pathways, ring A is formed from malonyl-CoA, and ring B is generated from 4-coumaroyl-CoA which is synthesized from phenylalanine via the shikimate pathway (Averesch and Krömer, 2018). Naringenin, an essential intermediate product, have neuroprotective and antioxidant properties. The other two consecutive products also have these kinds of properties. (Choi, 2012; Wedick et al., 2012; Liu et al., 2012; Raza et al., 2013; Hämäläinen et al., 2007)Especially, catechin, our final product, is a member of flavonoids which has significant effect on memory diseases and cardiovascular diseases.
Fig5: Metabolic pathway
What we do:
We use genes from nine different plants to produce two plasmids of Saccharomyces cerevisiae mainly by Golden Gate Assembly, Gibson Assembly and yeast homologous recombination to produce the flavonoids we need. The first plasmid is designed to produce naringenin. We use genes RtTAL, Pc4CL, GbCHS, MsCHI as coding sequence of this plasmid. The second plasmid is designed to produce catechin. We use genes CrCPR, CsF3H, GhF3'H, AaDFR, DuLAR as coding sequence of this plasmid. After these conversions, the bread would become useful to improve old people’s cognitive abilities.
Reference:
1.Alara, O. R., Abdurahman, N. H., and Ukaegbu, C. I. (2018a). Soxhlet extraction of phenolic compounds from Vernonia cinereal leaves and its antioxidant activity. J. Appl. Res. Med. Aromat. Plants 11, 12–17. doi: 10.1016/j.jarmap.2018.07.003
2.Averesch, N. J., and Krömer, J. O. (2018). Metabolic engineering of the shikimate pathway for production of aromatics and derived compounds-present and future strain construction strategies. Front. Bioeng. Biotechnol. 6:32. doi: 10. 3389/fbioe.2018.00032
3.Butelli, E.; Titta, L.; Giorgio, M.; Mock, H.-P.; Matros, A.; Peterek, S.; Schijlen, E. G. W. M.; Hall, R. D.; Bovy, A. G.; Luo, J.; Martin, C., Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnol. 2008, 26, 1301.
4.Choi, E.M., 2012. Liquiritigenin isolated from Glycyrrhiza uralensis stimulates osteoblast function in osteoblastic MC3T3-E1 cells. Int. Immunopharmacol. 12, 139–143. http://dx.doi.org/10.1016/j.intimp.2011.11.003.
5.Hämäläinen, M., Nieminen, R., Vuorela, P., Heinonen, M., Moilanen, E., 2007. Anti-inflammatory effects of flavonoids: genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and NF-jB activations, whereas flavone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-jB activation along with their inhibitory effect on iNOS expression and NO production in activated macrophages. Mediators Inflamm. http://dx.doi.org/10.1155/2007/45673
6.Liu, Y., Xie, S., Wang, Y., Luo, K., Wang, Y., Cai, Y., 2012. Liquiritigenin inhibits tumor growth and vascularization in a mouse model of HeLa cells. Molecules 17, 7206–7216. http://dx.doi.org/10.3390/molecules17067206
7.Raza, S.S., Khan, M.M., Ahmad, A., Ashafaq, M., Islam, F., Wagner, A.P., Safhi, M.M., Islam, F., 2013. Neuroprotective effect of naringenin is mediated through suppression of NF-kB signaling pathway in experimental stroke. Neuroscience 230, 157–171. http://dx.doi.org/10.1016/j.neuroscience.2012.10.041
8.Wedick, N.M., Pan, A., Cassidy, A., Rimm, E.B., Sampson, L., Rosner, B., Willett, W., Hu, F.B., Sun, Q., Van Dam, R.M., 2012. Dietary flavonoid intakes and risk of type 2 diabetes in US men and women. Am. J. Clin. Nutr. 95, 925–933. http://dx.doi. org/10.3945/ajcn.111.028894
9.Zhao, S., et al., Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering. Metab. Eng. (2014), http://dx.doi.org/10.1016/j.ymben.2014.12.002
10.The information about subjective cognitive decline is from https://www.cdc.gov/aging/data/subjective-cognitive-decline-brief.html
11.The key facts about aging population are from https://www.who.int/news-room/fact-sheets/detail/ageing-and-health