Resurrecting essential amino acid biosynthesis in mammalian cells
- NYU Langone Health, United States
- Columbia University, United States
- New York University, United States
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.
Article and author information
Author details
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
- Ivan Topisirovic, Jewish General Hospital, Canada
Version history
- Preprint posted: August 3, 2021 (view preprint)
- Received: August 6, 2021
- Accepted: September 26, 2022
- Accepted Manuscript published: September 27, 2022 (version 1)
- 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.
Metrics
-
- 3,300
- views
-
- 553
- downloads
-
- 4
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Resurrecting essential amino acid biosynthesis in mammalian cells
eLife 11:e72847.
https://doi.org/10.7554/eLife.72847
Further reading
-
- Cell Biology
High-altitude polycythemia (HAPC) affects individuals living at high altitudes, characterized by increased red blood cells (RBCs) production in response to hypoxic conditions. The exact mechanisms behind HAPC are not fully understood. We utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings indicate that HH significantly boosts erythropoiesis, leading to erythrocytosis and splenic changes, including initial contraction to splenomegaly over 14 days. A notable decrease in red pulp macrophages (RPMs) in the spleen, essential for RBCs processing, was observed, correlating with increased iron release and signs of ferroptosis. Prolonged exposure to hypoxia further exacerbated these effects, mirrored in human peripheral blood mononuclear cells. Single-cell sequencing showed a marked reduction in macrophage populations, affecting the spleen’s ability to clear RBCs and contributing to splenomegaly. Our findings suggest splenic ferroptosis contributes to decreased RPMs, affecting erythrophagocytosis and potentially fostering continuous RBCs production in HAPC. These insights could guide the development of targeted therapies for HAPC, emphasizing the importance of splenic macrophages in disease pathology.
-
- Cell Biology
Mapping proteins in and associated with the Golgi apparatus reveals how this cellular compartment emerges in budding yeast and progresses over time.
