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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription–translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors.

Details

Title
Cell-Free Protein Expression by a Reconstituted Transcription–Translation System Energized by Sugar Catabolism
Author
Sato, Gaku  VIAFID ORCID Logo  ; Miyazawa, Shintaro; Doi, Nobuhide; Fujiwara, Kei  VIAFID ORCID Logo 
First page
2956
Publication year
2024
Publication date
2024
Publisher
MDPI AG
e-ISSN
14203049
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
3079208259
Copyright
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.