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System-level studies of a cell-free transcription-translation platform for metabolic engineering

By Yong Y Wu, Hirokazu Sato, Hongjun Huang, Stephanie J. Culler, Julia Khandurina, Harish Nagarajan, Tae Hoon Yang, Stephen Van Dien, Richard M. Murray

Posted 03 Aug 2017
bioRxiv DOI: 10.1101/172007

Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.

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