RT Journal Article
SR Electronic
T1 Prototyping 1,4-butanediol (BDO) biosynthesis pathway in a cell-free transcription-translation (TX-TL) system
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 017814
DO 10.1101/017814
A1 Yong Y. Wu
A1 Stephanie Culler
A1 Julia Khandurina
A1 Stephen Van Dien
A1 Richard M. Murray
YR 2015
UL http://biorxiv.org/content/early/2015/04/09/017814.abstract
AB 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.