Abstract

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. 13C-tracing revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate in these cells. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at a consistent doubling time of 3.2 days. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

Data availability

Sequencing data generated for this study is deposited in the NCBI SRA at accession number PRJNA742028. Source data files have been provided for Figure 1 - figure supplement 1, Figure 1 - figure supplement 2D, Figure 2, Figure 2 - figure supplement 3, Figure 2 - figure supplement 4B, Figure 2 - figure supplement 5, Figure 2 - figure supplement 6, Figure 3, and Figure 3 - figure supplement 1, Figure 4, Figure 4 - figure supplement 1, Figure 5, and Figure 5 - figure supplement 1.

The following data sets were generated

Article and author information

Author details

  1. Julie Trolle

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2497-3531
  2. Ross M McBee

    Department of Biological Sciences, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  3. Andrew Kaufman

    Department of Systems Biology, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
  4. Sudarshan Pinglay

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  5. Henri Berger

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  6. Sergei German

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  7. Liyuan Liu

    Department of Systems Biology, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
  8. Michael J Shen

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  9. Xinyi Guo

    Department of Biology, New York University, New York, United States
    Competing interests
    No competing interests declared.
  10. J Andrew Martin

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  11. Michael E Pacold

    Department of Radiation Oncology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3688-2378
  12. Drew R Jones

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    No competing interests declared.
  13. Jef D Boeke

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    Competing interests
    Jef D Boeke, is a Founder and Director of CDI Labs, Inc., a Founder of Neochromosome, Inc, a Founder and SAB member of ReOpen Diagnostics, and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5322-4946
  14. Harris H Wang

    Department of Systems Biology, Columbia University, Columbia, United States
    For correspondence
    hw2429@columbia.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2164-4318

Funding

Defense Advanced Research Projects Agency (HR011-17-2-0041)

  • Jef D Boeke
  • Harris H Wang

National Human Genome Research Institute (RM1 HG009491)

  • Jef D Boeke

National Science Foundation (MCB-1453219)

  • Harris H Wang

Burroughs Wellcome Fund (PATH1016691)

  • Harris H Wang

Irma T. Hirschl Trust

  • Harris H Wang

Dean's Fellowship from the Graduate School of Arts and Sciences of Columbia University

  • Ross M McBee

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ivan Topisirovic, Jewish General Hospital, Canada

Version history

  1. Preprint posted: August 3, 2021 (view preprint)
  2. Received: August 6, 2021
  3. Accepted: September 26, 2022
  4. Accepted Manuscript published: September 27, 2022 (version 1)
  5. Version of Record published: October 13, 2022 (version 2)

Copyright

© 2022, Trolle et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Julie Trolle
  2. Ross M McBee
  3. Andrew Kaufman
  4. Sudarshan Pinglay
  5. Henri Berger
  6. Sergei German
  7. Liyuan Liu
  8. Michael J Shen
  9. Xinyi Guo
  10. J Andrew Martin
  11. Michael E Pacold
  12. Drew R Jones
  13. Jef D Boeke
  14. Harris H Wang
(2022)
Resurrecting essential amino acid biosynthesis in mammalian cells
eLife 11:e72847.
https://doi.org/10.7554/eLife.72847

Share this article

https://doi.org/10.7554/eLife.72847

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