Prototyping 1,4-butanediol (BDO) biosynthesis pathway in a cell-free transcription-translation (TX-TL) system
Abstract
Current methods for assembling metabolic pathways require a process of repeated trial and error and have a long design-build-test cycle. Further, it remains a challenge to precisely tune enzyme expression levels for maximizing target metabolite production. Recently it was shown that a cell-free transcriptional-translation system (TX-TL) can be used to rapidly prototype novel complex biocircuits as well as metabolic pathways. TX-TL systems allow protein expression from multiple DNA pieces, opening up the possibility of modulating concentrations of DNA encoding individual pathway enzymes and testing the related effect on metabolite production. In this work, we demonstrate TX-TL as a platform for exploring the design space of metabolic pathways using a 1,4-BDO biosynthesis pathway as an example. Using TX-TL, we verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as a limiting step of the 1,4-BDO pathway. We further tested combinations of various enzyme expression levels and found increasing downstream enzyme expression levels improved 1,4-BDO production.
Additional Information
The copyright holder for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. April 6, 2015, Submitted to 2015 Synthetic Biology: Engineering, Evolution & Design (SEED) Conference. We thank Genomatica for providing the materials used in this work, including plasmids encoding enzymes for the 1,4-BDO biosynthesis pathway and strain ECKh-422. We thank Nathan Dalleska and the Environmental Analysis Center for the support and assistance using GC/MS. This material is based upon work supported in part by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program; contract number HR0011-12-C-0065 (DARPA/CMO). Y.Y.W is supported by NIH/NRSA Training Grant 5 T32 GM07616. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.Attached Files
Submitted - 017814.full.pdf
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Additional details
- Eprint ID
- 57034
- Resolver ID
- CaltechAUTHORS:20150428-075140716
- DARPA/MTO Living Foundries program
- HR0011-12-C-0065
- NIH/NRSA Training Grant
- 5 T32 GM07616
- Created
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2015-04-28Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field