@article{62500cac0253453c807b8d41fce2b6fa,
title = "In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design",
abstract = "The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l−1. We expect iPROBE to accelerate design–build–test cycles for industrial biotechnology. [Figure not available: see fulltext.]",
author = "Karim, {Ashty S.} and Dudley, {Quentin M.} and Alex Juminaga and Yongbo Yuan and Crowe, {Samantha A.} and Heggestad, {Jacob T.} and Shivani Garg and Tanus Abdalla and Grubbe, {William S.} and Rasor, {Blake J.} and Coar, {David N.} and Maria Torculas and Michael Krein and Liew, {Fung Min (Eric)} and Amy Quattlebaum and Jensen, {Rasmus O.} and Stuart, {Jeffrey A.} and Simpson, {Sean D.} and Michael K{\"o}pke and Jewett, {Michael C.}",
note = "Funding Information: We thank A. M. Mueller, R. T. Tappel, W. Allen, L. Tran and S. D. Brown (LanzaTech) for conversations regarding this work. In addition, we thank C. Reynolds (Lockheed Martin) for conversations on the design of experiments using neural networks. This work is supported by the US Department of Energy, Office of Biological and Environmental Research in the Department of Environment Office of Science under award number DE-SC0018249. M.C.J. gratefully acknowledges the David and Lucile Packard Foundation and the Camille Dreyfus Teacher–Scholar Program. We also thank the following investors in LanzaTech{\textquoteright}s technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the New Zealand Superannuation Fund, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China and Suncor.",
year = "2020",
month = aug,
day = "1",
doi = "10.1038/s41589-020-0559-0",
language = "English (US)",
volume = "16",
pages = "912--919",
journal = "Nature Chemical Biology",
issn = "1552-4450",
publisher = "Nature Publishing Group",
number = "8",
}