Description

1. Background: Why Obesity?

1.1 Inspiration

At our school canteen, fried food is always the most popular snack during lunch break, with students often being seen coming and going with a bag of greasy, freshly fried chicken or french fries in their hands. Among these students, obesity and overweight problems have aready occurred, but according to a survey we conducted, the students are not aware of the potential dangers of obesity and are still sticking to their unhealty diet habits. Our teacher noticed this problem, and raised his concerns about us developing obesity. Long-term fast food intake and consequent obese problems have chronically afflicted him, he suffered from fatty liver diseass, inconveniences in daily life and is apprehensive about future implications. To make the matter worse, multiple weight-loss methods he tried out were all to no avail.

Further research suggest that obesity can lead to many physical and psychological problems. Physically, obesity is associated with several comorbidities, including cardiovascular disease, type 2 diabetes, sleep apnea, osteoarthritis, and several forms of cancer. Whereas psychologically, problems such as depression, eating disorders, substance abuse and emotional neglect have a correlation with obesity. This is an area worthy of in-depth study.

1.2 About Obesity

Excess body weight is the sixth most important risk factor. Obesity plays a relevant pathophysiological role in the development of health problems, arising as a result of the complex interaction of genetic, nutritional, and metabolic factors. Due to the role of adipose tissue in lipid and glucose metabolism, and low-grade inflammation, it is necessary to classify obesity based on body fat composition and distribution, rather than the simple increase of body weight, and the Body Mass Index.

1.2.1 Prevalence of Obesity

1.2.2 The Causes of Obesity

After investigation, we found several major factors that lead to obesity. It is worth mentioning that individual educational attainment, community median household income and community-built environment characteristics all have an impact on the incidence of obesity.

In addition to these, food access, transportation, advances in the work environment and communication technology, social class, economic stress, sleeping quality, marriage, physical activity, and workplace settings have the possibility to influence the incidence of obesity. Taken together, the occurrence of obesity is influenced by multiple factors from many aspects of life.

1.3 Current Solution

Current solutions to obesity can be divided into 3 categories: lifestyle and dietary modification, drug therapy and surgical interventions. However, each of these treatments have undesirable setbacks.

	Basic Information	Setbacks	Reference
Life Style and Dietary Modification	Mainly includes calories restriction, dietary supplements and engaging in physical activities to reach a negative energy	Difficult to maintain Relapse of obesity Low weight-loss efficacy	[1] Dalle Grave, R., Calugi, S. & El Ghoch, M. Lifestyle modification in the management of obesity: achievements and challenges. Eat Weight Disord 18, 339–349 (2013). [2] Soeliman FA, Azadbakht L. Weight loss maintenance: A review on dietary related strategies. J Res Med Sci. 2014 Mar;19(3):268-75.
Drug Therapy	Relatively efficient compared with Life style and dietary modification 5 classes of pharmacological methods: Orlistat, phentermine/ topiramate, lorcaserin, naltrexone/ bupropion, liraglutide Therapies act in blocking the digestion an absorption of triglycerides, suppressing appetite and improving satiety to reduce energy intake, and improving catabolism	Side effects such as nausea, head aches, insomnia and sometimes impairment of memory, attention and language, elevated blood pressure and gastrointestinal symptoms Low weight loss efficacy High cost	[1] Tak YJ, Lee SY. Long-Term Efficacy and Safety of Anti-Obesity Treatment: Where Do We Stand? Curr Obes Rep. 2021 Mar;10(1):14-30. [2] Williams DM, Nawaz A, Evans M. Drug Therapy in Obesity: A Review of Current and Emerging Treatments. Diabetes Therapy : Research, Treatment and Education of Diabetes and Related Disorders. 2020 Jun;11(6):1199-1216. [3] Muller, T. D., Clemmensen, C., Finan, B., DiMarchi, R. D. & Tschop, M. H. Anti-obesity therapy: from rainbow pills to polyagonists. Pharmacol. Rev. 70, 712–746 (2018).
Surgical Interventions	Laparoscopic adjustable gastric banding(LAGB) Liposuction Effective anti-obesity treatment with significant weight loss after surgery	Surgical risks and complications Anastomotic leak Anaesthetic complications Relapse	[1] Williams DM, Nawaz A, Evans M. Drug Therapy in Obesity: A Review of Current and Emerging Treatments. Diabetes Therapy : Research, Treatment and Education of Diabetes and Related Disorders. 2020 Jun;11(6):1199-1216.

Considering the above mentioned prevalence of obesity, its alarming consequences and the dismerit of current solutions, we aim to propose a solution to the problem. The rapid development and the promising application market of synthetic biology inspired us to resort to this method to tackle obesity.

But how are we going to apply synthetic biological methods?

In recent years, increasing evidence have shown that gut microtobia have critical influences on human body’s metabolism and energy balance. Gut microtobia relies on the food residues in the gut and produces multiple substances that could interact with human body. In terms of the relationship with obesity, studies have found that gut microbiota affects the host’s energy absorption, central appetite and fat storage, and therefore influences the weight gain and energy absorption of the host. Thus, gut microbiota has been regarded as a potential target of obesity.

Along with that, applying genetically engineering probiotics strands such as E.coli Nissle1917 and Saccharomyces boulardii to treat specific diseases has showed promising prospects. Not only can these genetically-modified strands bring probiotic benefits to human body, they can also specifically targeting diseases by influencing the composition of gut microbiota. Besides, compared to surgical and pharmacological methods, such biotherapeutic treatment decreases the cost and has significantly lower risks of harm to patients. This inspired us to construct engineered probiotics that could not only bring health benefits as a part of the gut microbiota, but also offer solutions to obesity.

2. Our Goal

We aim to construct a genetically modified E.coli Nissle 1917 strand which can absorb and metabolize ingested long chain fatty acid in human's small intestine, reduce the host's fat intake, and thus contribute to weight loss.

3.Our Design

3.1 Our Host Organism

We have chosen E.coli Nissle 1917, a common chassis for microbe, as our host organism. EcN is the only non-pathogenic strand of E.coli with probiotic properties. EcN had multiple fitness factors which impede the growth of opportunistic pathogens, interact with the intestinal epithelium to stimulate anti-inflammatory activities and restore and maintain intestinal barrier function.

Apart from this, the transcriptional and translational control of gene expression of EcN is heavily investigated, and there is wealthy knowledge of its metabolic circuits, which makes it a desirable candidate for genetic engineering.

3.2 The Integrate System (Including the Genes)

Our system comprise of 2 parts. System1 is responsible for the absorption and metabolism of LCFA, while System2 function as a kill switch that ensures biocontainment.

3.2.1 System 1: the LCFA Absorption and Metabolism System

1) Components and Functions

Our Product:

We constructed system1 to overexpress FadD and FadL in the E.coli cell.

FadL: FadL is the long chain fatty acid transport protein in E.coli.

FadL locates in the outer membrane, when bound with LCFA, FadL undergoes a conformational change which expose the transport channel and facilitate LCFA transport into the periplasmic space.

FadD: apart from FadL, the import of LCFA in e.coli is also dependent on FadD.

FadD is the only FACS (fatty acid Acetyl-CoA synthase) with a broad chain length specificity. FadD is responsible for the activation of exogenous LCFA for beta-oxidation and also regulates the transcription factor FadR.

FACS catalyzes the formation of fatty acyl CoA in 2 steps:

1. Fatty acid + ATP → Fatty acyl-AMP + PPi
2. Fatty acyl-AMP + CoA → Fatty acyl-CoA + AMP

2) As mentioned above, the LCFA import system in E.coli mainly depends on FadL and FadD. Within the bacterial cell, the LCFA transport is unidirectional because the imported FA becomes metabolically trapped by esterification with CoA, and is described as vectorial acylation.

1. LCFA binds to FadL, with conformational change, LCFA traverse the outer membrane via FadL.
2. LCFA is protonated in the periplasmic space, than then partition into the inner membrane through diffusion.
3. Inside the inner membrane, FadD activates LCFA to CoA thioesters.
4. After activation, LCFA go through beta-oxidation and provides respiratory substrate.

3.2.2 System 2: the Double Kill-switch System

1) Components and Functions

We used the biosafety design of the program of team Bielefeld Germany in 2013 for reference as our kill switch system. The biosafety design contains two parts.

The first part comprises of a L-rhamnose inducible promoter pRha (BBa_K914003 ), a positively regulated promoter that induces the expression of the genes downstream in the presence of L-Rhamnose, and genes encoding for alanine racemase(alr) ( BBa_K1172901) and the repressor araC.

The second part contains a promoter regulated by the above mentioned repressor pBAD and modified genes encoding RNase Barnase (BBa_K1172904), where the genes responsible for the extracellular translocation of RNase Barnase is deleted, allowing Barnase to catalyze the cleavage of bacterial ssRNA only intracellularlly.

2) Working Mechanism

1. In a confined system, such as the gut, with constant supply of L-rhamnose, the expression of alanine racemase and the repressor AraC is induced, converting L-alanine to D-alanine, a major component of the bacterial cell wall. Repressed by AraC, the RNase Barnase will not be expressed, and the bacteria functions normally within the gut.
2. After being excreted with feces, L-rhamnose would be gradually consumed. Thus the expression of alanine racemase is inhibited, and the bacterial cell would lack D-alanine the cross-linkage of cell wall peptidoglycan would be damaged, gives the bacterial cell a bacteriolytic characteristic and compromise cell division. Besides, without the repressor AraC, genes in the second part that code for RNase Barnase would be expressed, leading to rapid cell death.

This system functions as a double kill switch where lack of D-alanine and RNase Barnase could both contribute to rapid cell death when necessary, and is rather resistant to mutations.

But what if the plasmid is lost? To tackle this, we adjusted the genome DNA of our genetically-engineered probiotics by deleting the genome sequence of the above mentioned genes. Consequently, the bacterial cell would heavily depend on the plasmid gene that codes for the alanine racemase to live. If the plasmid is lost during the process, the bacterial cell would lyse.

4. Reference

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2. Black PN, DiRusso CC. Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification. Microbiol Mol Biol Rev. 2003 Sep;67(3):454-72, table of contents. doi: 10.1128/MMBR.67.3.454-472.2003. PMID: 12966144; PMCID: PMC193871.
3. Charbonneau, M.R., Isabella, V.M., Li, N. et al. Developing a new class of engineered live bacterial therapeutics to treat human diseases. Nat Commun 11, 1738 (2020). https://doi.org/10.1038/s41467-020-15508-1
4. Dalle Grave, R., Calugi, S. & El Ghoch, M. Lifestyle modification in the management of obesity: achievements and challenges. Eat Weight Disord 18, 339–349 (2013).
5. De Lorenzo, A., Soldati, L., Sarlo, F., Calvani, M., Di Lorenzo, N., & Di Renzo, L. (2016). New obesity classification criteria as a tool for bariatric surgery indication. World journal of gastroenterology, 22(2), 681–703. Mazloom, K., Siddiqi, I., & Covasa, M. (2019). Probiotics: how effective are they in the fight against obesity?. Nutrients, 11(2), 258.
6. Muller, T. D., Clemmensen, C., Finan, B., DiMarchi, R. D. & Tschop, M. H. Anti-obesity therapy: from rainbow pills to polyagonists. Pharmacol. Rev. 70, 712–746 (2018).
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9. Sarwer, D. B., & Polonsky, H. M. (2016). The Psychosocial Burden of Obesity. Endocrinology and metabolism clinics of North America, 45(3), 677–688.
10. Soeliman FA, Azadbakht L. Weight loss maintenance: A review on dietary related strategies. J Res Med Sci. 2014 Mar;19(3):268-75.
11. Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic. Microbial Ecology in Health and Disease, 21, 122 - 158.
12. Tak YJ, Lee SY. Long-Term Efficacy and Safety of Anti-Obesity Treatment: Where Do We Stand? Curr Obes Rep. 2021 Mar;10(1):14-30.
13. Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031 (2006).
14. Liu BN, Liu XT, Liang ZH, Wang JH. Gut microbiota in obesity. World J Gastroenterol. 2021 Jul 7;27(25):3837-3850. doi: 10.3748/wjg.v27.i25.3837. PMID: 34321848; PMCID: PMC8291023.
15. Weimar James D.,DiRusso,Concetta C.,Delio Raymond, Black Paul N. Functional Role of Fatty Acyl-Coenzyme A Synthetase in the Transmembrane Movement and Activation of Exogenous Long-chain Fatty Acids. Journal of Biological Chemistry. 2002 Aug;277(33)29369-29376. doi:10.1074/jbc.M107022200.
16. Williams DM, Nawaz A, Evans M. Drug Therapy in Obesity: A Review of Current and Emerging Treatments. Diabetes Therapy : Research, Treatment and Education of Diabetes and Related Disorders. 2020 Jun;11(6):1199-1216.
17. https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Biosafety-System_araCtive