Resurrecting essential amino acid biosynthesis in mammalian cells
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Curated by eLife
Evaluation Summary:
In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. 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 costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. 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. 13 C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. 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.
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Evaluation Summary:
In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.
(This preprint has been reviewed by eLife. We …
Evaluation Summary:
In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In this study, Trolle et al set out to investigate the impact of reintroduction of essential amino acid biosynthetic pathways into mammalian cells. To this end, they employed an elegant synthetic genomic approach to enable Chinese Hamster Ovary cells to endogenously produce methionine, threonine, isoleucine, and valine. Notwithstanding that attempts to functionalize biosynthesis of methionine, threonine and isoleucine were not successful, reintroduction of valine biosynthetic pathway rescued survival of Chinese Hamster Ovary cells deprived of valine. Moreover, the authors provide evidence that global mRNA abundance profiles in valine biosynthesis-proficient Chinese Hamster Ovary cells mirror those observed upon recovery from valine starvation. Collectively, these pioneering studies suggest potential for the …
Reviewer #1 (Public Review):
In this study, Trolle et al set out to investigate the impact of reintroduction of essential amino acid biosynthetic pathways into mammalian cells. To this end, they employed an elegant synthetic genomic approach to enable Chinese Hamster Ovary cells to endogenously produce methionine, threonine, isoleucine, and valine. Notwithstanding that attempts to functionalize biosynthesis of methionine, threonine and isoleucine were not successful, reintroduction of valine biosynthetic pathway rescued survival of Chinese Hamster Ovary cells deprived of valine. Moreover, the authors provide evidence that global mRNA abundance profiles in valine biosynthesis-proficient Chinese Hamster Ovary cells mirror those observed upon recovery from valine starvation. Collectively, these pioneering studies suggest potential for the functionalization of biosynthesis of essential amino acid in mammalian cells. Based on this, it was found that this study is of broad interest to a variety of research fields including synthetic biology, biotechnology, and biochemistry.
Strengths: This study incorporates a very elegant synthetic genomic approach to address a long-standing gap in knowledge related to exploring the ability of mammalian cells to tolerate restoration of essential amino acid biosynthesis. It was highly appreciated that this is one of the pioneering attempts to address this question. For the most part, the data were robust and supportive of the author's tenets. Finally, demonstration that Chinese Hamster Ovary cells can be rendered prototrophic for valine may open many interesting avenues in the domains of synthetic biology and biotechnology, with potential long-term applications in medicine.
Weaknesses: Relatively modest rescue of proliferation of valine-producing Chinese Hamster Ovary cells in valine-free media, apparent reduction in de novo valine synthesis during propagation of the cells and some technical issues pertinent to potential utilization of valine from breakdown of serum proteins were considered as the weaknesses of the study. Furthermore, it was thought that further molecular characterization of valine-prototrophic Chinese Hamster Ovary cells may be warranted.
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Reviewer #2 (Public Review):
In this manuscript, Trolle and McBee et al. looked to introduce biosynthetic pathways into Chinese Hamster Ovary (CHO) cell lines. In these efforts, the authors initially set out to generate a CHO cell line capable of endogenously producing methionine, threonine, isoleucine, and valine. Ultimately, the authors demonstrate partial restoration of the valine biosynthetic pathway but were unsuccessful in restoring the other EAA pathways. Based on the data presented, the authors offer convincing functional (cell viability), metabolomic, and proteomic evidence indicating valine biosynthesis was at least partially restored.
While the current study does demonstrate partial complement of the valine biosynthetic pathway, it is important to consider whether these results fully address the authors' main claims that this …
Reviewer #2 (Public Review):
In this manuscript, Trolle and McBee et al. looked to introduce biosynthetic pathways into Chinese Hamster Ovary (CHO) cell lines. In these efforts, the authors initially set out to generate a CHO cell line capable of endogenously producing methionine, threonine, isoleucine, and valine. Ultimately, the authors demonstrate partial restoration of the valine biosynthetic pathway but were unsuccessful in restoring the other EAA pathways. Based on the data presented, the authors offer convincing functional (cell viability), metabolomic, and proteomic evidence indicating valine biosynthesis was at least partially restored.
While the current study does demonstrate partial complement of the valine biosynthetic pathway, it is important to consider whether these results fully address the authors' main claims that this work both "represents a successful example of metazoan EAA biosynthesis restoration" and "demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways." To successfully substantiate this claim, one would expect convincing evidence indicating the engineered cell line could maintain a long-term homeostatic (i.e. "healthy") physiological state in the absence of valine supplementation.
Notwithstanding more minor concerns, this study's greatest limitation is reflected in the way by which the experimental design was adjusted to emphasize a successful outcome. These adjustments are apparent throughout the manuscript, such as employing a "conditioned-medium" feeding regiment, in which only 50% of the medium was changed out every 48 hours, and assessing transcriptional profiles of the engineered cells only at early timepoints (4 and 48hrs).
For a successful proof-of-concept, the engineered cell line should ideally exhibit a somewhat consistent doubling time over multiple passages with limited evidence of metabolic stress, even at later passages. Note, the observed doubling time would not necessarily be equivalent to cells cultured in valine replete medium, but the doubling time should be relatively stable. However, the authors report an average doubling time of 5.3 days over the first 24 days and an average doubling time of 21 days in the following 25 days. Reformatting the data to reflect doubling time at every passage would presumably show even more striking differences between early and later timepoints.
These data suggest that metabolic flux of the heterologous pathway is not sufficient to achieve an overall homeostatic state. Alternatively, it is also possible that metabolic inefficiencies are causing pathway intermediates/ byproducts to build up and negatively impact cell physiology. Both hypotheses would be quite straightforward to test, the first by comparing transcriptional profiles of the engineered cells at early (<24 days) and late (>24 days) timepoints, and the second by quantifying levels of metabolic intermediates in the engineered pathway over time.
Taken together, the results put forth in this manuscript suggest the authors were marginally successful in introducing a valine biosynthetic pathway into CHO cells, but fall short of demonstrating a robust, self-sustaining engineered cell line under reasonable culture conditions. This proof-of-concept could certainly be achieved by building on the preliminary findings reported in this study, either by further optimizing pMTIV expression strategies or testing enzymes in the valine biosynthetic pathway present in other organisms.
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Reviewer #3 (Public Review):
During evolution, eukaryotes lost the biosynthetic pathways that are responsible for the production of 9 amino acids. In this study, Wang et al successfully reintroduce the fully functional biosynthesis of these 9 amino acids back into mammalian cells. To accomplish this task, Wang et al had to introduce, into mammalian cells, >40 genes and reconstruct pathways that are naturally functional only in fungi plants, and bacteria. The entire pathway was synthesized de novo by commercial gene synthesis in 3 kilobase fragments and assembled in yeast. The work is a major bioengineering accomplishment that will serve for fundamental research into evolution and metabolism.
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