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Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets

Nature Biotechnology volume 23, pages 612–616 (2005)Cite this article

Abstract

Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.

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Figure 1: Systematic and combinatorial gene knockout target identification.
Figure 2: Visualization of the metabolic landscape.
Figure 3: Clustering analysis depicting the interaction of systematic and combinatorial targets.
Figure 4: Behavior of selected strains in optimized culturing conditions.

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Acknowledgements

We acknowledge financial support of this work by the DuPont-MIT Alliance. In particular, we would like to thank Wonchul Suh for providing the parental E. coli strain. We also thank Joel Moxley for providing thoughtful suggestions and Veronica Godoy for providing the initial phage stock.

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Author notes

  1. Kohei Miyaoku: On leave from Mitsubishi Chemical Corporation.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Room 56-469, Cambridge, 02139, Massachusetts, USA

    Hal Alper, Kohei Miyaoku & Gregory Stephanopoulos

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  1. Hal Alper
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Correspondence to Gregory Stephanopoulos.

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Supplementary information

Supplementary Fig. 1

Covariance analysis of systematic and combinatorial targets (PDF 113 kb)

Supplementary Table 1

Primer designs for gene knockout constructs (PDF 70 kb)

Supplementary Discussion (PDF 48 kb)

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Alper, H., Miyaoku, K. & Stephanopoulos, G. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23, 612–616 (2005). https://doi.org/10.1038/nbt1083

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