Abstract
Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. A possible application for such techniques is the bioremediation of natural water sources contaminated with small organic molecules (e.g., drugs and pesticides). A simple molecular system to actively accumulate and degrade pollutants could be a bionanoreactor composed of a liposome or polymersome scaffold combined with energizing- (e.g., light-driven proton pump), transporting- (e.g., proton-driven transporter) and degrading modules (e.g., enzyme). This work focuses on the engineering of a transport module specific for β-lactam antibiotics. We previously solved the crystal structure of a bacterial peptide transporter, which allowed us to improve the affinity for certain β-lactam antibiotics using structure-based mutagenesis combined with a bacterial uptake assay. We were able to identify specific mutations, which enhanced the affinity of the transporter for antibiotics containing certain structural features. Screening of potential compounds allowed for the identification of a β-lactam antibiotic ligand with relatively high affinity. Transport of antibiotics was evaluated using a solid-supported membrane electrophysiology assay. In summary, we have engineered a proton-driven β-lactam antibiotic translocation module, contributing to the growing toolset for bionanotechnological applications.
Highlights
Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature
One essential component needed for the development of such a system would be a transport module, which is able to accumulate the target molecule using energy provided by other components of the system
The specificity and affinity of the transporter YePEPT for certain β-lactam antibiotics was significantly improved using structure-based mutagenesis combined with functional characterization using an uptake assay in E. coli cells overexpressing the transporter
Summary
Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. Components and systems involved in biological processes are optimized or repurposed using engineering approaches to address specific challenges in a wide range of fields such as diagnostics, biotechnology and research[1,2]. This work focuses on the development of a molecular module from YePEPT, which is able to transport certain β-lactam antibiotics for application in a bioremediation system as described above (Fig. 1) and as a contribution for the growing toolset of modules, which can be used for molecular systems engineering. By combining structure-based mutagenesis with functional characterization, we were able to identify specific mutations, which enhance the affinity of YePEPT for certain β-lactam antibiotics into the low micromolar range. We could demonstrate transport of identified compounds and the suitability of the engineered YePEPT version as a β-lactam antibiotic translocation module
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