We decided to create probiotics that synthesize naringenin - a compound that belongs to flavonoids and has an antiprotozoal activity against Entamoeba histolytica. The synthesis of naringenin takes place in four reaction steps that are catalyzed by four enzymes: TAL (tyrosine ammonia-lyase from Herpetosiphon aurantiacus ), 4CL (4-coumarate-CoA ligase 2 from Glycine max ), CHS (chalcone synthase from Arabidopsis thaliana ), and CHI (chalcone isomerase from Medicago sativa ) . Although, a high concentration of the compound is needed to have the desired effect on our target amoeba species. Therefore we came up with an idea to increase the flavonoid synthesis by boosting the bottleneck reactions catalyzed by proteins 4CL and CHS by linking the two mentioned proteins together. Additionally, we were interested in checking whether the shorter distance between the active sites of the fused proteins provides higher efficiency of the reaction measured by the higher concentration of produced naringenin. For this purpose, we needed to determine what kind of structure this fusion protein complex would have, from which we could calculate the distance between active sites of the enzymes.
Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.
Usage and Biology
To find the most efficient way of naringenin biosynthesis we had to find enzymes of maximum activity and use them to construct naringenin production pathway in our target probiotic bacteria. To do so we relied on the article written by Mark S. Dunstan and colleagues  and decided to use fused 4CL from Glycine max and CHS from Arabidopsis thaliana. In addition to selecting the most promising enzymes, we also performed codon optimization on the gene sequences of interest. We also fused these two protein with flexible (GGGGS)2 linker in order to speed-up limiting rate reaction step.
Sequence and Features
- 10INCOMPATIBLE WITH RFCIllegal SpeI site found at 2893
- 12INCOMPATIBLE WITH RFCIllegal SpeI site found at 2893
- 21COMPATIBLE WITH RFC
- 23INCOMPATIBLE WITH RFCIllegal SpeI site found at 2893
- 25INCOMPATIBLE WITH RFCIllegal SpeI site found at 2893
Illegal NgoMIV site found at 2224
Illegal AgeI site found at 658
- 1000INCOMPATIBLE WITH RFCIllegal BsaI.rc site found at 1690
- Dunstan, M. S., Robinson, C. J., Jervis, A. J., Yan, C., Carbonell, P., Hollywood, K. A., ... & Scrutton, N. S. (2020). Engineering Escherichia coli towards de novo production of gatekeeper (2 S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol. Synthetic Biology, 5(1), ysaa012.