Team:TU Darmstadt/Project/Transport

Transport

The objective of group "Transport" is the integration of a terephtalic acid uptake system in Escherichia coli (E. coli). The uptake of TPA is crucial to produce high-value molecules into our host bacteria. TPA can only pass the membrane at low pH values, which adversely affects the growth of E. coli. Therefore, a suitable transport system is needed that operates under optimal growth conditions for E. coli.

Figure 1. The mechanism of terephtalalic acid (TPA ) uptake: Protein C binds the TPA and transfers it to the Proteins A and B, which transport the TPA across the inner membrane.

According to the data published by Sasoh et al.^[1] Comamonas testosteroni (C. testosteroni) is able to utilize TPA as the sole carbon and energy source. For that reason, we decided to isolate the putative TPA uptake System of C. testosteroni. This system belongs to the tripartite tricarboxylate transporters and consists of three subunits (A to C). The large subunit A is a with 11-12 alpha-helical transmembrane protein. It is acompanied by the small transmembrane subunit B which constis of 4-5 alpha-helical transmembrane protein. Subunit C is a specific periplasmic binding protein, which is moving freely in the periplasmic space and bonds to the AB units. (Fig.1) The function is similar to ABC transporters, however the sequences are unrelated. C. testosteroni features two different configurations of A and B proteins (A1 with 505 amino acids (aa) and B1 with 197 aa or A2 with 503 aa and B2 with 162 aa). The proteins are naturally unspecific and can transport different substrates.

We designed under the control of a Arabinose inducible promotor(AraC-Pbad) two putative terephtalate uptake operons. The intake of TPA was checked by photometry,gas chromatography-mass spectrometry (GC-MS) and energy dispersive X-ray spectroscopy (EDX). To determine the essential components and their combination for TPA transport into the cell, the genes were expressed in an overexpression strain E. coli C43(DE3). The structure characterisation was done by some bioinformatical tools like Protein Homology/anologY Recognition Engine V 2.0 (PHYRE2), I-TASSER servers, SignalIP 4.0 Server and TatP 1.0 Server. Additionaly, we developed a new method (ATS-method) to detect TPA with GC-MS.Futhermore, we characterized the AraC-Pbad regulation unit.

Detailed information on our approach is available in the Transport Labjournal. The next section contains information on the Metabolism step.

[1] Sasoh, M., E. Masai, et al. (2006). "Characterization of the terephthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 72(3): 1825-1832.

Results

• We created two Operons: Operon 1 and Operon 2 carrying our putative transport system reguled by an arabinose promoter
• We developed a new method to detect TPA-uptake in cells