Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains

Abstract

Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded E. coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered β-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely. We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.

The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology

“…A key reason for the use of microbes in biotech and research is their short generation time, and ALE allows for rapid improvement of this desirable trait. ALE has been applied to optimize the growth rate of various industrially relevant microbial species (Hong et al, 2011;LaCroix et al, 2014;Pfeifer et al, 2017;Yu et al, 2013), to ameliorate growth defects in engineered strains (Aguilar et al, 2018;Carroll and Marx, 2013;Radek et al, 2017;Thyer et al, 2018;Zelle et al, 2011), or to study how and why growth rate changes over the course of evolution (Barrick et al, 2009;Ferea et al, 1999;Lenski et al, 2015Lenski et al, , 1998Lenski et al, , 1991Lenski and Travisano, 1994;Weikert et al, 1997;Wong and Liao, 2009). ALE can significantly increase a strain's growth rate, even in frequently used culturing environments for which one might expect growth to already be close to optimal.…”

Therapeutic applications of genetic code expansion

“…Nevertheless, the positions that can be targeted on antibodies are limited. The incorporation of ncAAs using genetic expression systems like auxotrophic strain or selenocysteine have provided new insights [ 63 , 64 ].…”

Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases

“…Colorimetric [80], fluorescent [17,68,73,81] and luminescent [69,82] reporters have been adapted for medium/high-throughput screens of ncAA-acylating aaRS libraries. In all cases the reporter gene is translated with in-frame UAG codons and the cells with ncAA-acylating aaRS variants are collected based on the observable color, fluorescence or luminescence.…”

“…Translation systems to introduce ncAAs into proteins in vivo may cause different levels of cellular toxicity. While it is possible to develop customized strains that have evolved mechanisms to help them reduce this toxicity [81], for the purposes of comprehensive aaRS engineering a more forgiving platform would be desired. In addition to the inherent lack of toxic effects, in vitro evolution of proteins allows much larger focused libraries to be tested, because the library is not limited by the transformation efficiency.…”