In vitro transcription/translation-coupled DNA replication through the regeneration of 20 aminoacyl-tRNA synthetases

Hagino, Katsumi; Ichihashi, Norikazu

doi:10.51094/jxiv.53

##article.authors##

Hagino, Katsumi Graduate School of Arts and Science, The University of Tokyo
Ichihashi, Norikazu Graduate School of Arts and Science, The University of Tokyo

DOI:

https://doi.org/10.51094/jxiv.53

キーワード:

regeneration、 self-reproduction、 in vitro synthetic biology、 aminoacyl-tRNA synthetase、 artificial cell

抄録

The in vitro reconstruction of life-like self-reproducing systems is a major challenge in in vitro synthetic biology. Self-reproduction requires regeneration of all molecules involved in DNA replication, transcription, and translation. This study demonstrated the DNA replication and regeneration of major translation factors, 20 aminoacyl-tRNA synthetases (aaRS), in a reconstituted transcription/translation system (PURE system). First, we replicated each DNA that encode one of the 20 aaRSs through aaRS expression from the DNA (i.e., regeneration) by serial transfer experiments. Thereafter, we successively increased the number of aaRS genes and achieved simultaneous DNA replication and regeneration of all 20 aaRSs, which comprised approximately half the number of protein factors in the PURE system, except for ribosomes, by employing dialyzed reaction and sequence optimization. This study provides a step-by-step methodology for increasing the number of self-regenerative genes in self-reproducing artificial systems.

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引用文献

Szostak, J. W., Bartel, D. P. & Luisi, P. L. Synthesizing life. Nature 409, 387–390 (2001).

Forster, A. C. & Church, G. M. Towards synthesis of a minimal cell. Mol. Syst. Biol. 2, 45 (2006).

Damiano, L. & Stano, P. On the “Life-Likeness” of Synthetic Cells. Front. Bioeng. Biotechnol. 8, 1–6 (2020).

Cho, E. & Lu, Y. Compartmentalizing cell-free systems: Toward creating life-like artificial cells and beyond. ACS Synth. Biol. 9, 2881–2901 (2020).

Ivanov, I. et al. Bottom-Up Synthesis of Artificial Cells: Recent Highlights and Future Challenges. Annu. Rev. Chem. Biomol. Eng. 12, 287–308 (2021).

Wang, C., Yang, J. & Lu, Y. Modularize and Unite: Toward Creating a Functional Artificial Cell. Front. Mol. Biosci. 8, 1–14 (2021).

Laohakunakorn, N. et al. Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology. Front. Bioeng. Biotechnol. 8, 213 (2020).

Olivi, L. et al. Towards a synthetic cell cycle. Nat. Commun. 12, (2021).

Ichihashi, N. What can we learn from the construction of in vitro replication systems? Ann. N. Y. Acad. Sci. 1447, 144–156 (2019).

Forlin, M., Lentini, R. & Mansy, S. S. Cellular imitations. Curr. Opin. Chem. Biol. 16, 586–592 (2012).

Buddingh’, B. C. & van Hest, J. C. M. Artificial Cells: Synthetic Compartments with Life-like Functionality and Adaptivity. Acc. Chem. Res. 50, 769–777 (2017).

Yewdall, N. A., Mason, A. F. & Hest, J. C. M. Van. The hallmarks of living systems: Towards creating artificial cells. Interface Focus 8, 20180023 (2018).

Silverman, A. D., Karim, A. S. & Jewett, M. C. Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genet. 21, 151–170 (2020).

Noireaux, V. & Liu, A. P. The New Age of Cell-Free Biology. Annu. Rev. Biomed. Eng. 22, 51–77 (2020).

Blain, J. C. & Szostak, J. W. Progress Toward Synthetic Cells. http://dx.doi.org/10.1146/annurev-biochem-080411-124036 83, 615–640 (2014).

Gaut, N. J. & Adamala, K. P. Reconstituting Natural Cell Elements in Synthetic Cells. Adv. Biol. 5, 2000188 (2021).

Jia, T. Z., Wang, P. H., Niwa, T. & Mamajanov, I. Connecting primitive phase separation to biotechnology, synthetic biology, and engineering. J. Biosci. 2021 463 46, 1–28 (2021).

Stano, P. Gene Expression Inside Liposomes: From Early Studies to Current Protocols. Chem. – A Eur. J. 25, 7798–7814 (2019).

Bergquist, P. L., Siddiqui, S. & Sunna, A. Cell-Free Biocatalysis for the Production of Platform Chemicals. Front. Energy Res. 8, 193 (2020).

Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

Awai, T., Ichihashi, N. & Yomo, T. Activities of 20 aminoacyl-tRNA synthetases expressed in a reconstituted translation system in Escherichia coli. Biochem. Biophys. Reports 3, 140–143 (2015).

Li, J. et al. Cogenerating Synthetic Parts toward a Self-Replicating System. ACS Synth. Biol. 6, 1327–1336 (2017).

Shepherd, T. R. et al. De novodesign and synthesis of a 30-cistron translation-factor module. Nucleic Acids Res. 45, 10895–10905 (2017).

Libicher, K., Hornberger, R., Heymann, M. & Mutschler, H. In vitro self-replication and multicistronic expression of large synthetic genomes. Nat. Commun. 11, (2020).

Doerr, A., Foschepoth, D., Forster, A. C. & Danelon, C. In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity. Sci. Rep. 11, 1–12 (2021).

Libicher, K. & Mutschler, H. Probing self-regeneration of essential protein factors required for: In vitro translation activity by serial transfer. Chem. Commun. 56, 15426–15429 (2020).

Lavickova, B., Laohakunakorn, N. & Maerkl, S. J. A partially self-regenerating synthetic cell. Nat. Commun. 11, (2020).

Okauchi, H., Sakatani, Y., Otsuka, K. & Ichihashi, N. Minimization of Elements for Isothermal DNA Replication by an Evolutionary Approach. ACS Synth. Biol. 9, 1771–1780 (2020).

Sherman, J. M., Rogers, M. J. & Söll, D. Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation. Nucleic Acids Res. 20, 2847–2852 (1992).

Gene Frontier web site, 6 Tips about the template DNA Design. https://purefrex.genefrontier.com/resources/tech_notes/template-dna-design.html.

Kazuta, Y., Matsuura, T., Ichihashi, N. & Yomo, T. Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system. J. Biosci. Bioeng. 118, (2014).

Hibi, K. et al. Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs. Commun. Biol. 3, (2020).

Fujino, T., Tozaki, M. & Murakami, H. An Amino Acid-Swapped Genetic Code. ACS Synth. Biol. 9, 2703–2713 (2020).

Iwane, Y. et al. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nat. Chem. 8, 317–325 (2016).

Chen, J., Chen, M. & Zhu, T. F. Translating protein enzymes without aminoacyl-tRNA synthetases. Chem 7, (2021).

Calles, J., Justice, I., Brinkley, D., Garcia, A. & Endy, D. Fail-safe genetic codes designed to intrinsically contain engineered organisms. Nucleic Acids Res. 47, 10439–10451 (2019).

Endy, D. Experimental tests of functional molecular regeneration via a standard framework for coordinating synthetic cell building. bioRxiv 2021.03.03.433818 (2021).

Tamaru, D., Amikura, K., Shimizu, Y., Nierhaus, K. H. & Ueda, T. Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors. RNA 24, 1512–1519 (2018).

Shimojo, M. et al. In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli. Commun. Biol. 3, 142–142 (2020).

Miyachi, R., Shimizu, Y., Ichihashi, N. Jxiv 10.51094/jxiv.35 (2022) doi:DOI: https://doi.org/10.51094/jxiv.35.

Okauchi, H. & Ichihashi, N. Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System. ACS Synth. Biol. 10, 3507–3517 (2021).

Sakatani, Y., Yomo, T. & Ichihashi, N. Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination. Sci. Rep. 8, 13089 (2018).

Shimizu, Y., Kanamori, T. & Ueda, T. Protein synthesis by pure translation systems. Methods 36, 299–304 (2005).

Ito, Y., Kawama, T., Urabe, I. & Yomo, T. Evolution of an Arbitrary Sequence in Solubility. J. Mol. Evol. 58, 196–202 (2004).