The aim of this thesis will be to approach model bacterial systems (such as E. coli and Mycoplasma) that are used in minimal cell development research. The genomes of these model organisms have already been successculy reduced in the genome/proteome size and on the level of the information-carrying DNA. However, no study so far limited the number of the amino acids that compose the proteome. Such a reduction would lead to energetic savings and uncover whether cells could function with less than 20 amino
acids to compose their proteins. We have already learnt that proteins could work with a much smaller amino acid repertoire in vitro. This thesis will focus on whole biological systems using genome engineering and laboratory evolution. Initially, the dispensability of selected amino acids will be compared for the studied bacterial systems using starvation experiments. The amino acid that will be most dispensable will then be substituted in exemplary metabolic pathways using genome engineering technique and broad randomization schemes. This thesis will explore whether an extant biological system can cope with removal of one (or more) amino acid from its proteome, how significant mark it would leave and whether cells could adapt to it by forward evolution. 

Five relevant publications of the research group:

Tretyachenko V, Vymetal J, Neuwirthova T, Vondrasek J, Fujishima K, Hlouchova K*. (2022) Modern and prebiotic amino acids support distinct structural profiles in proteins. Open Biology 12:220040.

Giacobelli VG, Fujishima K, Lepsik M, Tretyachenko V, Kadava T, Bednarova L, Novak P, Hlouchova K*. (2022) In vitro evolution reveals non-cationic protein – RNA interaction mediated by metal ions. Molecular Biology and Evolution, msac032.

Makarov M, Meng J, Tretyachenko V, Srb P, Březinová A, Giacobelli VG, Bednárová L, Vondrášek J, Dunker AK* and Hlouchová K*. (2021) Enzyme catalysis prior to aromatic residues: reverse engineering of a dephosphoCoA kinase. Protein Science

Makarov M, Sanchez Rocha AC, Krystufek R, Cherepashuk I, Dzmitruk V, Charnavets T, Faustino AM, Lebl M, Fujishima K, Fried SD*, Hlouchova K*. (2023) Early selection of the amino acid alphabet was adaptively shaped by biophysical constraints of foldability. J Am Chem Soc. 145(9):5320-5329.

Heames B, Buchel F, Aubel M, Tretyachenko V, Loginov D, Novak P, Lange A, Bornberg- Bauer E*, Hlouchova K*. (2023) Experimental characterization of de novo proteins and their unevolved random-sequence counterparts. Nature Ecology & Evolution

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