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bioRxiv · 10.1101/172007

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

Wu, Y. Y.·Sato, H.·Huang, H.·Culler, S. J.·Khandurina, J.·Nagarajan, H.·Yang, T. H.·Van Dien, S.·Murray, R. M.
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Abstract

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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Wu, Y. Y., Sato, H., Huang, H., Culler, S. J., Khandurina, J., Nagarajan, H., Yang, T. H., Van Dien, S., Murray, R. M.. 2017-08-03. System-level studies of a cell-free transcription-translation platform for metabolic engineering. https://doi.org/10.1101/172007

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