Essential proteins as tools of selections for novel orthogonal tRNA/tRNA-amino acyl synthetase pairs

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Tilmann Kuenzl, Sonja Billerbeck, Michael Hoesl, Nediljko Budisa, Philippe Marliere, Sven Panke

ETH Zurich, Switzerland

Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. On the other hand they potentially make excellent selection systems as it is difficult to obtain false positives. An impediment to this effort is the lack of simple growth-based selection systems suitable for directed evolution approaches. As the genes are essential, producing knock-out strains is not possible and alternative strategies such as bleach out strains are tedious because they lead to situations where wild-type and variant genes co-exist and recombination confuses experimental outcomes. We established a simple strategy for genetic replacement of essential genes by a (library of) variant(s) during transformation1. A central element of the method is that the complementation vector carrying a copy of the wild-type form of the essential gene contains an I-SceI nuclease recognition site and can thus be rapidly and conditionally eliminated in the presence of an I-SceI nuclease-expressing helper plasmid. We applied the system to the engineering of the essential enzymes adenylate kinase (Adk), the essential chaperonin GroEL, and the glycerol-3-phosphate dehydrogenase GpsA in Escherichia coli to confirm its generality. We are currently expanding the system to explore the construction of selection strains for the design of novel orthogonal pyrrolysine aminoacyl-tRNA synthetase/tRNA (PylRS) pairs. By providing essential proteins with alternative, non-canonical amino acids, very powerful selection systems can be constructed and the selection system can be fortified against hitherto overlooked potential pitfalls of the screening process. This process will be coupled to efforts of developing pathways for the in vivo synthesis of the non-canonical amino acids to obtain an informationally expanded strain that is again autonomous in terms of amino acid synthesis.