Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.
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The authors thank D. Pitera for the pADSmut plasmid and W. Holtz for the BglBrick plasmids with the lacUV5 promoter and LacIQ removed. J.A.-G. thanks Fundacion Ramon Areces for his PostDoc fellowship. This work was part of the Department of Energy Joint BioEnergy Institute (http://www.jbei.org/) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.
J.D.K. has a financial interest in Amyris, Lygos and LS9.
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Dahl, R., Zhang, F., Alonso-Gutierrez, J. et al. Engineering dynamic pathway regulation using stress-response promoters. Nat Biotechnol 31, 1039–1046 (2013). https://doi.org/10.1038/nbt.2689
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