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Stabilized gene duplication enables long-term selection-free heterologous pathway expression

Nature Biotechnology volume 27, pages 760–765 (2009)Cite this article

Abstract

Engineering robust microbes for the biotech industry typically requires high-level, genetically stable expression of heterologous genes and pathways. Although plasmids have been used for this task, fundamental issues concerning their genetic stability have not been adequately addressed. Here we describe chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli. CIChE uses E. coli recA homologous recombination to evolve a chromosome with ∼40 consecutive copies of a recombinant pathway. Pathway copy number is stabilized by recA knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities. Comparison of CIChE-engineered strains with equivalent plasmids revealed that CIChE improved genetic stability approximately tenfold and growth phase–specific productivity approximately fourfold for a strain producing the high metabolic burden–biopolymer poly-3-hydroxybutyrate. We also increased the yield of the nutraceutical lycopene by 60%. CIChE should be applicable in many organisms, as it only requires having targeted genomic integration methods and a recA homolog.

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Figure 1: Chemically induced chromosomal evolution (CIChE) evolves the chromosome of a microorganism to produce many copies of a recombinant allele.
Figure 2: Allele segregation indicates that random distribution, not mutation rates, result in rapid productivity loss in plasmids.
Figure 3: In the absence of antibiotics, gene copy number in CIChE recA− cells is more stable than in plasmid-bearing or CIChE recA+ cells.
Figure 4: Gene copy number and yield in CIChE strains meet or exceed plasmid counterparts.
Figure 5: CIChE improves genetic stability.

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Acknowledgements

We would like to acknowledge National Science Foundation grant CBET-0730238 and the Singapore-MIT Alliance for funding. We thank H. Alper for cloning advice and J. Young for model calculations advice. Lysogens and transfer strains were kindly provided by D. Boyd and J. Beckwith (Harvard Medical School). BW26,547 was received from B. Sauer (Massachusetts Institute of Technology (MIT)). pAGL20 is a gift from A. Sinskey (MIT).

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  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Keith E J Tyo, Parayil Kumaran Ajikumar & Gregory Stephanopoulos

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  1. Keith E J Tyo
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  3. Gregory Stephanopoulos
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Contributions

K.E.J.T. designed the study, performed modeling, strain engineering and PHB evaluation. P.K.A. evaluated lycopene strains. G.S. supervised all work. K.E.J.T., P.K.A. and G.S. analyzed data and wrote paper.

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Correspondence to Gregory Stephanopoulos.

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Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Equations (PDF 661 kb)

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Tyo, K., Ajikumar, P. & Stephanopoulos, G. Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nat Biotechnol 27, 760–765 (2009). https://doi.org/10.1038/nbt.1555

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