DopaDoser: The Self-Regulating, L-DOPA-Producing Gut Bacteria

Fig 1 Administration of L-DOPA via bacteria in the patient's gut

iGEM Manchester-Graz’s objective as a team is to find a better way to treat and alleviate the symptoms of Parkinson’s disease (PD) through the use of synthetic biology to create a new auto-regulated expression system called DopaDoser. Degradation of dopaminergic neurons and therefore low levels of dopamine is the main cause of Parkinson’s disease (PD), for which the current treatment involves oral doses of L-DOPA (or levodopa), which unlike dopamine itself is able to cross the blood-brain-barrier. Within the brain L-DOPA is enzymatically converted into dopamine and therefore able to relieve many of the motor symptoms of PD.

Patients who cannot take the traditional treatments due to several conditions (intolerance or too high tolerance to oral L-DOPA) are treated alternatively with a so called Duodopa ® therapy. Duodopa is a special way for administration of L-DOPA (Levodopa ®) directly into the human jejunum through a PEG-PEJ tube. This approach allows for a continuous and stable dosage of L-DOPA over the whole day. However some major problems of this approach are high costs (around 700 $ per week plus costs for placement of the tube and aftercare) and wear out of the tube every 2 years. Also the system needs to be rinsed on a daily basis and during the night medication has to be taken oral anyways. The annual costs per patient are around 100.000 $.

Fig 2 dopamine and L-DOPA biosynthesis pathways

Our aim is to take the first steps in the development of a novel technology of drug delivery by developing self-regulating drug-producing bacterial strains that, in the future, could be incorporated into patients’ gut microflora to secrete medicines directly inside the body. To control the bacterial L-DOPA production, we plan to develop a multidimensional cell density dependent auto-regulation system that could also be used to control other multistep enzyme pathways.

The Manchester section of the team is working on L-DOPA and dopamine biosynthesis in E. coli BL21 and Nissle 1917 via various enzyme pathways. The focus is on three enzyme pathways, the main one being the conversion of L-tyrosine to L-DOPA via tyrosine hydroxylase and tyrosinase. In addition we are also synthesising dopamine in two different ways using aromatic amino acid decarboxylase, cytochrome P450 and transaminase. Although the primary goal would be to implement the L-DOPA synthesis within patients, we also aim to create a greener, more efficient way of the industrial synthesis of each of the above products using our modified bacteria. (Fig.2)

The Graz section is using two quorum sensing based mechanisms for an auto-regulated and time shifted consecutive protein expression, first demonstrated using fluorescent protein synthesis (Fig.3). The fluorescent protein could then be exchanged with genes for the L- DOPA production such that at low cell density levels tyrosine synthesis will be channeled. After a certain biomass is reached L-DOPA production will be produced in a uniformly continuous manner. Alongside the use of fluorescent proteins for the characterization of our regulatory system, we also try to further characterize and improve already existing reporter genes of the biobrick registry. BBa_K1670003 is an improved variant of BBa_K1362461, where two silent mutations were introduced to delete two HindIII sites and a new ribosome binding site as well as a new promoter were added. BBa_K1670001 is an improved version of BBa_E0020, where also a new ribosome binding site and an EsaR/I regulated promoter was added.

Fig 3 Fluorescent protein synthesis dependent on different levels of cell density

Looking to the future our system could be further utilized to activate suicide genes in E.coli to avoid possible overgrowth of the native intestinal flora, which we aim to show proof of concept in both strains common to academic research as well as probiotics, specifically BL21 and Nissle 1917 respectively. Even though we cannot regulate the proliferation of our engineered strain yet, it allows us to provide an outlook of a possible application as a self-regulating drug dispensing system in the GI tract, which may have clinical applications in the future.

Throughout the course of the DopaDoser project we also aim to shape our actions in accordance to the opinions of academics, charities, industry leaders and the public. This includes what implications DopaDoser will have for patients both economically and in terms of quality of life and whether the real world implementation of this technology is feasible. We will also compare ethical opinions throughout outreach about our project and synthetic biology in general across the two countries our team spans and assess this in a wider sociological context. We feel as a team that the issues and human practices surrounding our project are just as important as the project itself and as such we endeavour to tackle a wide selection of concerns.