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Prototyping a valinomycin biosynthesis pathway within a cell-free transcription-translation (TX-TL) system

Zhou, Tiffany (2016) Prototyping a valinomycin biosynthesis pathway within a cell-free transcription-translation (TX-TL) system. . (Submitted)

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Many natural metabolites have antibacterial, antiviral, or anticancer effects and can be developed into new drugs. However, working with the microorganisms that produce these products can be challenging since they are not as well characterized as a model organism like Escherichia coli. In this paper, we investigate the potential for a cell-free transcription-translation (TX-TL) system to provide a rapid prototyping platform for characterizing new genetic pathways. We use the valinomycin biosynthesis pathway as a test case, and we show successful heterologous expression of the heterodimeric valinomycin synthetase (VlmSyn, Vlm1: 374 kDa and Vlm2: 284 kDa) from Streptomyces tsusimaensis within the TX-TL system. Using LC-MS analysis, we find that valinomycin is produced at low but detectable levels, even when only one out of the three basic precursors is fed into the system. Our work represents another step towards applying cell-free biosynthesis to the discovery and characterization of new natural products.

Item Type:Report or Paper (Discussion Paper)
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Additional Information:The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint first posted online Dec. 4, 2016. We would like to thank Prof. Dr. Peter Neubauer (TU Berlin) and Dr. Jian Li (Northwestern University) for providing us with their pVlm1, pVlm2, and sfp plasmids, their E. coli BJJ01 strain, and their helpful comments regarding the valinomycin biosynthesis pathway. We are also thankful for Dr. Nathan Dalleska (Caltech) for facilitating the LC-MS analysis, Yong Wu (Caltech) and Clare Hayes (Caltech) for providing input on techniques used in lab, and Dr. Richard Murray (Caltech) for providing valuable advice throughout the course of the project and the permission to conduct research in his laboratory. Funding for this project was provided by the Caltech Grubstake program.
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Caltech Grubstake ProgramUNSPECIFIED
Record Number:CaltechAUTHORS:20170320-150910879
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:75255
Deposited By: Tony Diaz
Deposited On:20 Mar 2017 22:18
Last Modified:15 Nov 2021 16:32

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