It has been more than 50 years since Robert Guthrie suggested the first newborn screening program for phenylketonuria (PKU). To this day, avoiding foods containing high levels of phenylalanine remains the most important treatment factor for people with PKU. Almost every variety of food rich in protein must be avoided, this includes dairy products, meat, fish or chocolate. Despite the fact that strict diet is an effective way to prevent clinical presentation of PKU, it is still very difficult for the patients to implement it into everyday life. Only about half of the patients with PKU in Lithuania report to be following the required strict diet. Even those reporting to be following the treatment regime have an IQ that is about one half a standard deviation lower than their unaffected siblings or the population average. Moreover, people with PKU cannot get enough nutrients using conventional food sources and about 70-80 % protein intake must be comprised of low-phenylalanine supplements. There are situations in which getting the required mixture is problematic. For example, after Lithuania declared its independence in 1990, its supply of protein supplements was cut off due to an economic blockade. As a result, people born that year have clinical manifestations of PKU because proper treatment was not available. Finally, protein supplements are costly: in Lithuania a child under 18 years old is compensated for about 12 000 euros (13 160 US dollars) annualy.
Lithuania has some of the most experienced specialists in the region working both with newborn screening and the treatment of PKU. In 1975, Lithuania became the first country in the former Soviet Union to have started a newborn screening program for PKU. It has been a substantial achievement for that time period, as not even Moscow had a newborn screening program of its own. For comparison, our closest neighbours, Latvia and Estonia, started their screening program for PKU in the early 90s. Lithuania’s active involvement in the new treatment options for PKU and other genetic diseases was one of the reasons we chose it as our topic.
The first of our two approaches for probiotic treatment of PKU is based on the enzyme, called phenylalanine ammonia lyase (PAL). Naturally, PAL enzyme is found mostly in plants and fungi and is involved in the response of various stimuli, such as pathogens, light, temperature.
The main function of PAL is to break down L-phenylalanine to trans-cinnamic acid and ammonia (picture). Both of these compounds are easily degraded to other metabolic molecules and have no toxic effect. Mainly because of this, PAL enzyme may serve as possible substitution to phenylalanine hydroxylase (PAH), the main phenylalanine processing enzyme in human cells, which is inactive in PKU cases.
A small number of researchers have previously reported a potential PKU treatment using PAL enzyme, however, none of these studies got as far as clinical trial stage, mainly because of the therapy design or low expression levels. Most of the researches considered injecting an active PAL enzyme directly into the bloodstream of the patient. Even though this is definitely the quickest approach, it causes numerous immunological problems. In addition, it is also difficult to maintain enzymatic PAL activity up to the certain level in the bloodstream.
Rather than using naked enzyme directly in the bloodstream of the patient, we considered creating a probiotic bacteria to deliver PAL to the intestine, where food is digested and most of the amino acids are transported to the bloodstream. This way enzyme itself is protected from most of the environmental factors and can steadily maintain its activity. At the same time, because probiotic bacteria will be found in the intestine, not in the bloodstream of the patient, it does not cause immune response.
In our project we used PAL enzyme sequence derived from Anabaena variabilis, cyanobacteria. We chose PAL enzyme naturally found in bacteria because it is more likely to maintain its function in our model organism E. coli. Our team used PAL biobrick, previously created by Stanford-Brown 2015 iGEM team (K1692004), and managed to improve it by adding different kind of tags to its sequence (K1983000) and K1983001.
It is rather simple, but effective and brilliant approach. Our newly engineered probiotic bacteria with expressed PAL activity would take care of excess phenylalanine in the human gut after every meal. Protected by bacteria from the harsh environment of the gastrointestinal tract, PAL easily maintains its enzymatic activity (see results).
Following PAL activity results, we considered additional ways to make the system even more efficient. We found phenylalanine-specific permease gene pheP (hyperlink), normally found in Escherichia coli, which main function is phenylalanine uptake in the cell, and cloned it into our cells with PAL enzyme. Significantly bigger absorbtion of phenylalanine into the cell results in the substantial increase of its break down, which is the goal of our project. You can read more about system efficiency modulation on our result page) and system activity under real life conditions on our demonstration page.
Our second approach is based on the central dogma of molecular biology. It is outstandingly straightforward, yet reasonably compelling. The idea is simple: at the usual conditions in the bacterial cell, most of phenylalanine molecules are either degraded or used for protein synthesis. Consequently, in order to increase the number of used phenylalanine molecules, we need to utilize more of it by incorporating them to the protein.
Therefore, we created synthetic genes, which sequence is constructed from as many phenylalanine codons, as possible, starting from 5 per cent and rising up to as much as 21 per cent of all the amino acids in the protein (see how we chose proteins and did the design). Phenylalanine molecules, which approach the probiotic bacterial cell in the human intestine, will be combined into the hydrophobic protein. High expression rates of these 'polyphe proteins' (i.e. proteins, exceptionally rich in the latter amino acid) results in greater uptake of phenylalanine molecules from the cell cytosol than in regular conditions (see our results).
Polyphe protein approach is completely novel and unconventional, and, in addition, it has a huge advantage over every possible treatment for PKU done before. Biobrick device with polyphe protein and strong promoter would result in high expression levels of our proteins in the cell. PAL (or other enzymes in that matter) are much more sensitive to cell environment, and even small changes in pH, temperature or other factors can cause a significant change in enzyme activity. Polyphe proteins, on the other hand, will be synthesized by any means.
As we proceeded with our idea, we realized that one of the limiting factors of the system might be the inefficient amount of tRNAs, bearing phenylalanine in the cell (tRNA-Phe). On that account, we created tRNA-Phe biobrick (K1983010) and combined it together with two kinds of promoters – constitutive (K1983011) and naturally found in E. coli DH10B strain (K1983012). We integrated tRNA-Phe together with polyphe proteins into the cells to make one working unit. In theory, bigger expression of tRNA-Phe consequently leads to more RNA molecules, carrying amino acids and therefore greater protein expression as well as increased system activity. However, our experiments did not confirm this hypothesis. Due to this, we can claim that tRNA-Phe expression is either not a limiting factor or the difference between system with and without it was too little to determine (see our results and demonstration).
To develop our project even further beyond the lab bench, we have a few objectives. It is particularly important to reach the highest expression levels possible for both of the approaches – PAL enzyme and polyphe proteins. Also, both of these systems (PAL and polyphe proteins), working in cooperation in one cell, could undoubdtedly become the most succesful approach and reach the maximum effectiveness. This would maximize the amount of phenylalanine uptaken from the human intestine into the probiotic bacteria. Read more about maximum system efficiency on our modelling page.
Another significant issue is safety and system regulation. One of the main reasons, why such probiotic bacteria might be prohibited to use, is continous fear of genetically modified organisms (GMOs) and their manipulation. In the view of the fact that our bacteria, in theory, would be used directly by people and could possibly cause medical issues, it is of great importance to regulate its life cycle and activity. Last year Vilnius-Lithuania iGEM 2015 team created a bacterial timer as a control mechanism. Combining this method together with post-transcriptional control, such as aptamers and riboswitches, could serve as a potential regulatory unit. We performed an extensive literature research on this topic at the early stages of the project, however, this kind of research would require a substantial amount of time and resources.
Since the final product of our project is a probiotic, is it our concern to consider the most suitable chassis organism for the matter. Even though we conducted our experiments in conventional model organism E. coli, we reviewed Bacillus subtilis 168 strain for experimenting in the laboratory as well as B. subtilis HU58 strain for the actual use of product. B. subtilis HU58 is already acknowledged as a strain, widely common in the human intestine, and has all the probiotic cell properties. In addition, it has a vast advantage over E. coli, as it forms spores, which can be dried down and placed in the pill.
Due to the fact, that we would like to use B. subtilis spores, we thought that it might be a good idea to induce their growth before they get into the intestine. This way our bacteria would start working immediately after getting into the intestine. Prebiotics, which would be used to initiate spore growth, in theory would be packed inside of the capsule and surrounded by B. subtilis spores. The idea is a double capsule – one inside another. Before taking the capsule of probiotics, it would be necessary to squeeze it. After that, inner capsule breaks apart and interaction between prebiotics and probiotics induce the growth of the bacteria.
The probiotic delivery method is also one of the things, that needs to be taken into the account. In collaboration with Oxford iGEM 2016 team, we conducted a survey (see the results of the survey on our human practices page) about how people see probiotics and what kind of drug delivery they would like to use. The survey showed that people prefer taking pharmaceutical products over food based products.
- Medical Genetics: An Integrated Approach, Schaefer, G. Bradley/ Thompson, James N., Jr., McGraw-Hill Professional Pub, 2013, ISBN 10: 0071664386
- Tam NKM, Uyen NQ, Hong HA, Duc LH, Hoa TT, Serra CR, Henriques AO, Cutting SM. The Intestinal Life Cycle of Bacillus subtilis and Close Relatives. Journal of Bacteriology. 2006, p. 2692–2700
- Fakhry S, Sorrentini I, Ricca E, De Felice M, Baccigalupi L. Characterization of spore forming Bacilli isolated from the human gastrointestinal tract. Journal of Applied Microbiology. 2008; 2178–2186
- Hong HA, Khaneja R, Tam NMK, Cazzato A, Tan S, Urdaci M, Brisson A, Gasbarrini A, Barnes I, Cutting SM. Bacillus subtilis isolated from the human gastrointestinal tract. Research in Microbiology. 2009; 134-143
- Permpoonpattana P, Hong HA, Khaneja R, Cutting SM. Evaluation of Bacillus subtilis strains as probiotics and their potential as a food ingredient. Beneficial Microbes. 2012; 3(2): 127-135