Overview

1. Inspiration

2. Background

3. Description

4. Goals

Inspiration

While brainstorming, our team found a shared interest in the gut microbiome due to its large diversity and varied composition of microbes and its interconnectedness with many other areas of human health. Due to the impact of the gut microbiome on nutrition and homeostasis, we decided that it would be an ideal lens through which to look at metabolic disorders. We looked at previous iGEM projects on rare metabolic disorders such as Phenylketonuria (PKU), Wilson's Disease, and Hemochromatosis that sparked our interest. We ultimately settled on Maple Syrup Urine Disease at the suggesion of one of our mentors due to its interesting pathophysiology and the presence of an unmet need due to a lack of an accessible and long-term treatment for the disease.

Background

In healthy individuals, branched chain amino acids (BCAAs) are metabolized through reverse transamination into their corresponding branched chain keto-acids (BCKAs) in skeletal muscle tissue, followed by the oxidative catabolism of excess BCKAs by the branched chain keto-acid dehydrogenase (BCKDH) complex in the liver. In classical MSUD, one or more subunits of the BCKDH complex is non-functional, resulting in the accumulation of BCAAs and their keto-acid derivatives to toxic levels. Depending on the exact mutation present, the effect of this can take several forms of severity, ranging from partial or intermittent function to complete dysfunction. If left untreated, MSUD results in progressive neurological damage and ultimately death. Treatment of the disease consists of strict dietary management to limit protein intake throughout the patient's lifetime. Dialysis, hemofiltration, and/or tube feeding may be required during severe episodes of metabolic decompensation, which can be triggered by physiological stress, regardless of diet or disease severity. Currently, the only permanent cure for MSUD is a liver transplant. However, as with all transplants, waiting time, access to a high level of medical care, and post-transplant complications pose significant barriers to widespread curative measures. Such an invasive solution may also not be desirable or accessible to individuals with less severe versions of the disease.

For the majority of individuals suffering from MSUD, the ability to control the diet is crucial for preventing irreversible neurological damage. However, such control may be difficult or costly to manage, and leaves limited room for error. This is the gap in MSUD research that our project aims to address.

Description

Our B. subtilis probiotic is engineered to:
1) Increase intracellular BCAA concentrations through constitutive expression of bcaP, a high-affinity BCAA importer, and knockout of azlC and azlD, both of which are BCAA exporters.
2) Increase intracellular BCAA breakdown through constitutive expression of ilvE and ilvK, responsible for the transamination of BCAAs into BCKAs, and constitutive expression of the bkd operon, responsible for oxidative decarboxylation of BCKAs.

We envision our probiotic acting as a non-invasive and convenient supplement for individuals affected by MSUD to help them achieve a greater flexibility in their diet. Future directions we hope to take include the incorporation of our probiotic into baby formula, as well as the colonization of the gut with our bacteria for a long-term treatment.

Why synthetic biology?

By taking advantage of the tools of synthetic biology to clone and transform genes into new circuits, we are able to repurpose the existing pathway of BCAA breakdown in bacteria to develop an easily-deliverable probiotic. Bacteria are also easily tuned to their surroundings, and making regulation of gene expression readily achievable to modulate protein expression during varying periods of gut BCAA levels. Similarly, using a recombinase switch, we are able to maintain the fitness of our probiotic bacteria by delaying the induction of gene expression until the probiotic is ready to be delivered or digested. Alternatively, using a BCAA biosensor, the bacteria would be able to autonomously turn on gene expression when the surrounding concentration of BCAAs reaches a high enough threshold.

Goals

Wet Lab

Our primary goal is to show that increasing expression of bcaP, ilvE/ilvK, and the bkd operon increases the uptake and breakdown of BCAAs from the environment. Additional considerations include adding regulatory elements such as a recombinase switch to prevent the induction of gene expression until the probiotic is ingested or cell density is sufficiently high, as well as self-regulatory elements such as a BCAA biosensor for further modulating gene expression.

Modeling

Our goal for our model is to develop a representation for the rate of consumption of BCAAs in the gut environment under various conditions and methods of gene regulation in order to predict the effectiveness of our probiotic in the context of an actual patient.

Human Practices

As MIT has a tradition of annual meetups with other iGEM teams, we hosted a gut microbiome meetup with other teams working on similar projects. We also met with various experts on metabolic diseases, as well as MSUD in particular; scientists at local biotechs working in synthetic biology; and other stakeholders personally affected by MSUD in order to develop a more practical and feasible solution.