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Prototyping 1,4-butanediol (BDO) biosynthesis pathway in a cell-free transcription-translation (TX-TL) system

Wu, Yong Y. and Culler, Stephanie and Khandurina, Julia and Van Dien, Stephen and Murray, Richard M. (2015) Prototyping 1,4-butanediol (BDO) biosynthesis pathway in a cell-free transcription-translation (TX-TL) system. . (Submitted)

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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.

Item Type:Report or Paper (Discussion Paper)
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Murray, Richard M.0000-0002-5785-7481
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.
Funding AgencyGrant Number
DARPA/MTO Living Foundries programHR0011-12-C-0065
NIH/NRSA Training Grant5 T32 GM07616
Subject Keywords:TX-TL, cell-free systems, metabolic pathway, and 1,4-butanediol
Record Number:CaltechAUTHORS:20150428-075140716
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Official Citation:Prototyping 1,4-butanediol (BDO) biosynthesis pathway in a cell-free transcription-translation (TX-TL) system Yong Y Wu , Stephanie Culler , Julia Khandurina , Stephen Van Dien , Richard M Murray bioRxiv doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57034
Deposited By: Tony Diaz
Deposited On:28 Apr 2015 19:13
Last Modified:03 Oct 2019 08:20

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