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Improving lycopene production in Escherichia coli by engineering metabolic control

Nature Biotechnology volume 18, pages 533–537 (2000)Cite this article

Abstract

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.

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Figure 1: Design strategy of the dynamic controller represented using the block diagram of control engineering.
Figure 2: Characterization of the dynamic controller.
Figure 3: Protein overexpression from multicopy plasmids with and without the dynamic controller.
Figure 4: Metabolic control engineering of lycopene production.
Figure 5: Dynamic control by ACP and the glnAp2 loop enhances lycopene production from E. coli.

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References

  1. Bailey, J.E. Toward a science of metabolic engineering. Science 252, 1668–1675 (1991).

    Article  CAS  Google Scholar 

  2. Guillouet, S., Rodal, A.A., An, G., Lessard, P.A. & Sinskey, A.J. Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production. Appl. Environ. Microbiol. 65, 3100–3107 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Kurland, C.G. & Dong, H. Bacterial growth inhibition by overproduction of protein. Mol. Microbiol. 21, 1–4 (1996).

    Article  CAS  Google Scholar 

  4. Sies, H. & Stahl, W. Lycopene: antioxidant and biological effects and its bioavailability in the human. Proc. Soc. Exp. Biol. Med. 218, 121–124 (1998).

    Article  CAS  Google Scholar 

  5. Giovannucci, E. Tomatoes, tomato-based products, lycopene and cancer: review of the epidemiological literature. J. Natl. Cancer Inst. 91, 317–331 (1999).

    Article  CAS  Google Scholar 

  6. Wang, C.-W., Oh, M.-K. & Liao, J.C. Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli. Biotechnol. Bioeng. 62, 235–241 (1999).

    Article  CAS  Google Scholar 

  7. Miura, Y. et al. Production of the carotenoids lycopene, β-carotene, and astaxanthin in the food yeast Candida utilis. Appl. Environ. Microbiol. 64, 1226–1229 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kajiwara, S., Fraser, P.D., Kondo, K. & Misawa, N. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem. J. 324, 421–426 (1997).

    Article  CAS  Google Scholar 

  9. Ruther, A., Misawa, N. & Sandmann, G. Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids. Appl. Microbiol. Biotechnol. 48, 162–167 (1997).

    Article  CAS  Google Scholar 

  10. Hoch, J.A. & Silhavy, T.J. Two-component signal transduction. (ASM Press, Washington, DC; 1995).

  11. Nyström, T. The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival. Mol. Microbiol. 12, 833–843 (1994).

    Article  Google Scholar 

  12. McCleary, W.R. & Stock, J.B. Acetyl phosphate and the activation of two-component response regulators. J. Biol. Chem. 269, 31567–31572 (1994).

    CAS  PubMed  Google Scholar 

  13. Stock, J.B., Surette, M., McCleary, W.R. & Stock, A.M. Signal transduction in bacterial chemotaxis. J. Biol. Chem. 267, 19753–19756 (1992).

    CAS  PubMed  Google Scholar 

  14. Kim, S.-K., Wilmes-Riesenberg, M.R. & Wanner, B.L. Involvement of the sensor kinase EnvZ in the in vivo activation of the response regulator PhoB by acetyl phosphate. Mol. Microbiol. 22, 135–147 (1996).

    Article  CAS  Google Scholar 

  15. Feng, J. et al. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J. Bacteriol. 174, 6061–6070 (1992).

    Article  CAS  Google Scholar 

  16. Reitzer, L.J. & Magasanik, B. Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc. Natl. Acad. Sci. USA 82, 1979–1983 (1985).

    Article  CAS  Google Scholar 

  17. Liu, J. & Magasanik, B. Activation of the dephosphorylation of nitrogen regulator I-phosphate of Escherichia coli. J. Bacteriol. 177, 926–931 (1995).

    Article  CAS  Google Scholar 

  18. Hwang, I., Thorgeirsson, T., Lee, J., Kustu, S. & Shin, Y.K. Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC. Proc. Natl. Acad. Sci. USA 96, 4880–4885 (1999).

    Article  CAS  Google Scholar 

  19. Patnaik, R., Roof, W.D., Young, R.F. & Liao, J.C. Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle. J. Bacteriol. 174, 7527–7532 (1992).

    Article  CAS  Google Scholar 

  20. Sprenger, G.A. et al. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc. Natl. Acad. Sci. USA 94, 12857–12862 (1997).

    Article  CAS  Google Scholar 

  21. Misawa, N. et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172, 6704–6712 (1990).

    Article  CAS  Google Scholar 

  22. Lerner, C.G. & Inouye, M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 18, 4631 (1990).

    Article  CAS  Google Scholar 

  23. Fürste, J.P. et al. Molecular cloning of the plasmid RP4 primase region in a multi-host range tacP expression vector. Gene 48, 119–131 (1986).

    Article  Google Scholar 

  24. Patnaik, R., Spitzer, R.G. & Liao, J.C. Pathway engineering for production of aromatics in Escherichia coli: confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities. Biotechnol. Bioeng. 46, 361–370 (1995).

    Article  CAS  Google Scholar 

  25. Patnaik, R. & Liao, J.C. Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield. Appl. Environ. Microbiol. 60, 3903–3908 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Dedhia, N., Richins, R., Mesina, A. & Chen, W. Improvement in recombinant protein production in ppGpp-deficient Escherichia coli. Biotechnol. Bioeng. 53, 379– (1997).

    Article  Google Scholar 

  27. Farmer, W.R. & Liao, J.C. Reduction of aerobic acetate production by Escherichia coli. Appl. Environ. Microbiol. 63, 3205–3210 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Möckel, B., Eggeling, L. & Sahm, H. Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis. Mol. Microbiol. 13, 833–842 (1993).

    Article  Google Scholar 

  29. Fussenegger, M., Schlatter, S., Bailey, J.E. & Mazur, X. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat. Biotechnol. 16, 468–472 (1998).

    Article  CAS  Google Scholar 

  30. Stewart, V. & Parales, J., Jr. Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12. J. Bacteriol. 170, 1589–1597 (1988).

    Article  CAS  Google Scholar 

  31. Silhavy, T.J., Berman, M.L. & Enquist, L.W. Experiments with gene fusions. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1984).

  32. Simons, R.W., Houman, F. & Kleckner, N. Improved single and multicopy lac -based cloning vectors for protein and operon fusions. Gene 53, 85–96 (1987).

    Article  CAS  Google Scholar 

  33. Draths, K.M. et al. Biocatalytic syntesis of aromatics from D-glucose: the role of transketolase. J. Am. Chem. Soc. 114, 3956–3962 (1992).

    Article  CAS  Google Scholar 

  34. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  35. Miller, J.H. A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1992).

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Acknowledgements

The authors gratefully acknowledge Chia-wei Wang of our laboratory for the construction of pCW9. This work was supported by the National Science Foundation (grant BES-9814097) and the US Department of Energy (DE-FG03-95ER20205).

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  1. Department of Chemical Engineering, University of California Los Angeles, Los Angeles, CA 90034

    William R. Farmer & James C. Liao

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Correspondence to James C. Liao.

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Farmer, W., Liao, J. Improving lycopene production in Escherichia coli by engineering metabolic control. Nat Biotechnol 18, 533–537 (2000). https://doi.org/10.1038/75398

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