Team:KCIS NewTaipei/Description

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What is Vitamin D?
Vitamin D, a fat soluble vitamin, exists in two forms: ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Humans can obtain vitamin D2 through consuming plant-based food sources such as mushrooms and soy milk; and, D3 through animal based products such as fatty fish or liver (Office of Dietary Supplements - Vitamin D, 2017). Vitamin D3 can also be synthesized by the human skin after exposure to ultraviolet B (UVB) light (280-315 nm) (Bordelon et al., 2009). UVB light from sun rays convert 7-dehydrocholesterol (7-DHC) in the skin into pre-vitamin D3, which is then isomerized into vitamin D3 (Wacker & Holick, 2013).

In the liver, both vitamin D2 and D3 are metabolized into 25(OH)D, the inactive form of vitamin D. 25(OH)D is then converted into its activated form, 1,25(OH)2D in the kidneys. The biological role of 1,25(OH)2D is mediated through the vitamin D receptor (VDR). VDR binds to the retinoid X receptor (RXR) to form a heterodimer, which then binds to its ligand 1,25(OH)2D to form 1,25(OH)2D-VDR complex. The ligand-bound VDR complex then regulates gene expression by binding to the vitamin D response elements (VDRE), a sequence in the promoter region of the target gene (Hossein-Nezhad et al., 2014).

Vitamin D plays a pleiotropic role in the body. Research has shown that vitamin D regulates calcium metabolism, the immune system, cardiovascular system, and others. For instance, in the immune system, 1,25(OH)2D induces the expression of the gene CAMP, CD14, FN1, TREM1, which are responsible for encoding proteins involved in immediate response after infection (Saponaro et al., 2020).
*Fig. 1 Overview of vitamin D metabolism in the human body
Vitamin D Deficiency: A Global Pandemic
Announced by the NIH (National Institute of Health) as a global pandemic, vitamin D deficiency (serum 25(OH)D level <50 nmol/L) has already affected over 1 billion individuals around the world (Ibrahim, et al 2020). Despite no obvious symptoms, around 50% of the global population has vitamin D insufficiency issues (serum 25(OH)D level of 50 ~ <75nmol/L)(Holick et al, 2021; Amrein, et al, 2021; Charoenngam et al., 2021).
Vitamin D World Map
*Fig. 2 Any levels of vitamin D below 75 nmol/L are considered insufficient. Most of the population lies between either 50-74 nmol/L or 25-49 nmol/L, both considered vitamin D insufficient (Interactive Maps on Vitamin D Level Worldwide, 2012).
Health professionals utilize serum 25(OH)D concentrations as the marker for assessing vitamin D status instead of serum 1,25(OH)2D concentrations since its supply is more plentiful in metabolites of vitamin D in human serum. Furthermore, 25(OH)D is also more stable compared to 1,25(OH)2D as it has a half-life of about 15 days while 1,25(OH)2D only has up to 15 hours, making 25(OH)D a better clinical candidate for determination of vitamin D status(Thacher & Clarke, 2011). However, while it is most widely used for clinical measurements, recent studies have suggested this method as unreliable since it is 1,25(OH)2D that interacts with Vitamin D receptors(VDR) and induces gene expression. Thus, the ratio of 1,25(OH)2D to 25(OH)D (“Vitamin D activation ratio”) makes a much better indication for determining vitamin D status in the human body (Thomas et al., 2020).

While there are several causes of deficiency, the most common reasons are the lack of exposure to sunlight and absorption of vitamin D from day-to-day diets (Holick, 2021). Without sufficient amounts of vitamin D, individuals start developing symptoms such as bone density loss, vulnerable immune system, and fatigue. Vitamin D deficiency also has strong associations to numerous chronic diseases, including bone metabolic disorders, tumors, and diabetes (Wang, et al 2017). In adults, vitamin D deficiency may worsen illnesses such as osteopenia, osteoporosis, and fractures. In children, lack of vitamin D can increase the chance of developing rickets, a rare bone disease that can cause bone pain, soft bones, and bone deformities (Vitamin D Deficiency, 2020; NHS Choices, 2018).
COVID-19 and Vitamin D
Known for regulating mineral metabolism, bone growth, and calcium absorption, vitamin D shares several strong bonds with biological actions during immunomodulation (Clarke, 2009). Recent studies even suggest possible associations between the biological actions of vitamin D and protection against COVID-19. This includes the immunomodulatory effects of innate and adaptive immune systems, reduction of viral entry in the kidney and lungs, as well as protective effects against possible COVID-19 associated kidney injuries (Charoenngam et al., 2021). Although vitamin D supplementation’s potential in combating severity and intensity of COVID-19 has not yet been confirmed, increasing amounts of scientific experimentations support the claim. With COVID-19 on the roll, increasing and maintaining vitamin D serum levels is without a doubt beneficial in minimizing the damage caused by this disease (Charoenngam et al., 2021).
Supplements and Medication
One of the most common solutions to combat vitamin D deficiency is by taking vitamin D supplements. For children, these supplements boost bone growth and muscle density. For adults, it improves both bone and mental health while combating prevalent issues like depression. This form of treatment, however, is a short-term solution. Also, since children often lack awareness towards the benefits of supplements/medication, convincing them to take supplements daily/regularly may prove to be a challenge in many households. Furthermore, with amnesia caused by diseases such as Alzheimer's, there is a high chance for adults/elders to forget to or over take their medication/supplements. If patients do not take their pills regularly, their serum 25(OH)D levels can easily drop below the normal range (30 ng/mL,) (Tello, 2016). (NatureWise, 2021)
Vitamin D Supplement
*Fig. 3 Photo of vitamin D supplement (NatureWise, 2021)
UV Light Therapy
Known as another alternative towards solving vitamin D deficiency, UVB light therapy utilizes UVB rays to penetrate the skin in order to increase conversion of 7-DHC into vitamin D3. During the treatment, patients enter a room equipped with UVB lamps. UVB rays will then initiate vitamin D metabolism, further increasing the patient's serum vitamin D level. Despite this therapy’s advantage in combating vitamin D deficiency, several dermatologists argue that at-home UVB light treatment should not be implemented due to its perceived high risk, including skin cancer, loss of skin elasticity, and eye problems. Also, incorrect usage of UVB light lamps may induce gene damage and mutation (Bhutani & Liao, 2010).
UV Light Therapy
*Fig. 4 Photo of UV light therapy (Phototherapy | UV Light Therapy - Toronto Dermatology Centre, 2021)
To create a better solution for combating vitamin D deficiency, we engineer butyrate-producing bacteria.
Target Genes & Protein
Acetyl-CoA is the pathway that most butyrate-producing bacteria species use to produce butyrate. As a result, it is the preferred pathway for our project (Vital et al., 2014).
Butyrate-producing Pathway Chart
*Fig. 5 Butyrate-producing pathways in metagenomic data from samples of 15 healthy individuals are calculated and demonstrated as a percentage of total bacterial genomes theoretically expressing a pathway. The different color bars represent individual butyrate-producing pathways. The orange, blue, pink, and grey represent acetyl-CoA, glutarate, 4-aminobutyrate, and lysine pathways respectively. The 15 samples analyzed in the research are demonstrated by the box plot. (Vital et al., 2014)
Gene in the Biological System
According to past research that assessed the butyrate metabolic pathway by measurement of intracellular pathway intermediates in recombinant Escherichia coli, the pool size measurement and thermodynamic analysis showed the step converting Crotonyl-CoA to butyryl-CoA as the rate-limiting step of the acetyl-CoA butyrate synthesis pathway (Fischer et al., 2010). As a result, we identified the bcd-etfAB gene, which encodes for butyryl-CoA dehydrogenase, as our gene of interest.

The experiment also proves that the step converting acetoacetyl-CoA to (3S)-3-hydroxybutyryl-CoA is the rate-limiting step among the first three steps of the acetyl-CoA butyrate-producing pathway (from acetyl-CoA to Crotonyl-CoA) (Fischer et al., 2010). We, therefore, also target the hbd gene, which encodes for beta-hydroxybutyryl-CoA dehydrogenase.
Butyrate Butyrate-producing Pathaway
*Fig. 6 Different colors represent the different butyrate-producing pathways. Acetyl-CoA is the butyrate-producing pathway (orange) our project investigates. (Vital et al., 2014)
Butyrate is a short-chain fatty acid synthesized by probiotics in human guts. It increases VDR (Vitamin D Receptor) expression in a time and dose dependent manner through the activity of the TGFβ pathway (Apprato, 2020).

Compared to the half-life of 25(OH)D (15 days), the half-life of the activated form, 1,25(OH)2D (15 hours) is relatively short. In the human body, 1,25(OH)2D is degraded and metabolized often before it is even utilized to exert downstream effects on certain genes, wasting activated vitamin D.

The upregulation of VDR increases the binding activity between VDR and the 1,25(OH)2D that would have been wasted otherwise. Increased binding activity will allow for the expression of certain health-related genes , thus helping the human body perform processes such as the regulation of the immune system and calcium homeostasis.

In order to create a better long-term solution to the problem of Vitamin D deficiency, we aim to enhance the butyrate-producing ability of probiotics.
To achieve our goal, we insert the target genes into the target probiotic. The hbd/ bcd-etfAB genes would result in the expression of beta-hydroxybutyryl-CoA dehydrogenase/ butyryl-CoA dehydrogenase, therefore increasing the rate of butyrate production.

Finally, we incorporate the resulting butyrate producing bacteria in the fermented food of countries worldwide to put our idea into practice.
Butyrate-producing Process
*Fig.7 Overview of how our engineered butyrate producing bacteria in the fermented food starter increase VDR expression and binding activity between 1,25(OH)2D and VDR.
When we first started as an iGEM team, our team started brainstorming ideas about what kind of topics we would feel passionate about. Looking at different research papers and recent studies about a wide range of topics and taking inspiration from other iGEM teams, we found a paper about “golden rice,” a genetically modified food. This Golden rice project from 1999, although had several ethical oppositions, still managed to help children in around 16 countries combat vitamin A deficiency (Genetically Modified Organisms: The “Golden Rice” Debate). Therefore, after deep consideration, our team also decided to dedicate our project to something just as great, to help out others. And so, we came up with the inspiration to do a project also related to vitamins, which came to be vitamin D.
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