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Designing an Escherichia coli Strain for Phenylalanine Overproduction by Metabolic Engineering

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Abstract

The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG fbr performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications. Knocking in the tktA gene under a tyrR promoter and knocking out pykF further increased phenylalanine production demonstrating that the supply of precursor via PEP and E4P is also a rate-limiting step. Finally, we tested the role of global regulators on this deregulated pathway and found that Fis overexpression helps in both enhancing and sustaining the flux through this pathway. This work opens up the possibility of using global regulators in synergy with pathway-specific modifications to enhance product yields.

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Acknowledgements

We thank Dr. Jean Pierre Hernalsteens, Belgium for providing plasmids pKD3, pKD4 pKD46 and pCP20. NT wishes to acknowledge Council of Scientific and Industrial Research, Government of India, and DST-Purse grants for providing the fellowship. The DBT-BUILDER Project (BT/PR5006/INF/22/153/2012) is acknowledged for instrument facility. The author also wishes to acknowledge the GLP and CIF facility in SBT-JNU.

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Authors and Affiliations

  1. School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

    Neetu Tyagi, Deepti Saini, Richa Guleria & Krishna Jyoti Mukherjee

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  1. Neetu Tyagi
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  2. Deepti Saini
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  3. Richa Guleria
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  4. Krishna Jyoti Mukherjee
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Correspondence to Krishna Jyoti Mukherjee.

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Tyagi, N., Saini, D., Guleria, R. et al. Designing an Escherichia coli Strain for Phenylalanine Overproduction by Metabolic Engineering. Mol Biotechnol 59, 168–178 (2017). https://doi.org/10.1007/s12033-017-9999-5

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  • DOI: https://doi.org/10.1007/s12033-017-9999-5

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