Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set

Tomoshige Fujino, Ryogo Sonoda, Taito Higashinagata, Emi Mishiro-Sato, Keiko Kano and Hiroshi Murakami ()
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Tomoshige Fujino: Nagoya University
Ryogo Sonoda: Nagoya University
Taito Higashinagata: Nagoya University
Emi Mishiro-Sato: Nagoya University
Keiko Kano: Nagoya University
Hiroshi Murakami: Nagoya University

Nature Communications, 2024, vol. 15, issue 1, 1-10

Abstract: Abstract The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors.

Date: 2024
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DOI: 10.1038/s41467-024-48056-z

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