The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments1,2. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales3. Although in vitro and directed evolution methods4,5,6,7,8,9 have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
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We are grateful to J. Jacobson for his insights and advice throughout this work. We thank D. Court for his insights and sharing strain DY330, N. Reppas for advice and sharing strain EcNR2, F. X. Cunningham for sharing pAC-LYC, and B. H. Sterling for assistance in constructing the EcFI5 strain. We also thank M. Jewett, J. Aach, D. Bang, S. Kosuri and members of the Church laboratory for advice and discussions. We thank the NSF, DOE, DARPA, the Wyss Institute for Biologically Inspired Engineering and training fellowships from the NIH and NDSEG (H.H.W.) for supporting this research.
Author Contributions H.H.W., F.J.I. and G.M.C. conceived the study jointly with P.A.C.; H.H.W. and F.J.I. designed and performed experiments with assistance from P.A.C., Z.Z.S., G.X. and C.R.F.; H.H.W. and F.J.I. wrote the manuscript; G.M.C. supervised all aspects of the study.
we wish to disclose that three authors (G.M.C., H.H.W, F.J.I.) have a pending patent application whose value may be affected by the publication of this paper. G.M.C. also discloses various associations with companies as outlined at http://arep.med.harvard.edu/gmc/tech.html.
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Wang, H., Isaacs, F., Carr, P. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009). https://doi.org/10.1038/nature08187
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