Team:Aachen/Description

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Project Description

S. cerevisiae

E. coli

Synthetase

Saccharomyces cerevisiae - targeting serine

Escherichia coli - targeting tyrosine

Escherichia coli is widely used in synthetic biology. It offers the advantage of being a comparatively simple and well-understood model organism while being easy to handle in the laboratory environment. Also, an expansion of the genetic code has already been successfully implemented in E. coli multiple times [1]–[4] by introducing an orthogonal tRNA/aminoacyl-synthetase pair.
Therefore, working in E. coli is an obvious choice.

Due to a limited range of tRNA/aminoacyl-synthetase pairs for non-canonical amino acids in general and especially for those that act orthogonally in E. coli, photocaging serine in the active site of subtilisin E with DMNB-serine is currently not possible. Hence, another strategy is needed to produce temporarily inactive proteases. This part of the project focuses on utilizing the maturation process of subtilisin E.

Figure 1: maturation process of subtilisin E

Subtilisin E is an alkaline serine protease found in Bacillus subtilis that has to autoprocess itself to become functional. At first, the enzyme exists as a precursor, namely the pre-pro-subtilisin. The pre-sequence serves as a recognition sequence for secretion across the cytoplasmic membrane and is cleaved off in the course of the process. The pro-peptide acts as an intramolecular chaperone and facilitates the folding of the protease. Folding is essential for the activity of an enzyme. Still, the maturation process of Subtilisin E is not completed, as the pro-peptide covers the substrate binding site and inhibits activity. However, enough proteolytic activity is achieved to autoprocess the IMC-domain and therefore cleave off the pro-peptide. Yet, the C-terminal end of the pro-peptide continues to block the substrate binding site. After the degradation of the pro-peptide, the substrate-binding site is cleared and the protease becomes effectively active. [5]

This mechanism can be used to implement a novel inactivation method.

O-(2-Nitrobenzyl)-L-tyrosine (abbr.: ONBY) is a derivate of the canonical amino acid tyrosine. It carries a photo-labile protection group that can be cleaved off by irradiation with UV-light (365nm, [6]).

Figure 2: structural formula of O-(2-Nitrobenzyl)-L-tyrosine

Synthetase - incorporating DMNBS in E. coli