Abstract
Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed, and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. The evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source.
Key points
• Adaptive evolution of E. coli enables biosynthesis of all amino acids from methanol.
• Overexpression of threonine biosynthesis pathway improves methanol assimilation.
• Methanol-dependent growth is seen in minimal media with homoserine as co-substrate.
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Data availability
All strains generated in this study will be made available upon request.
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Acknowledgments
The authors would like to thank Dr. Hannes Link for kindly providing the dysregulated E. coli strain, thrA*, from which the pThr* plasmid was constructed from, used in this study.
Funding
This work was supported by the Advanced Research Projects Agency-Energy (ARPA-E) Reducing Emissions using Methanotrophic Organisms for Transportation Energy (REMOTE) program (DE-AR0000432).
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All authors contributed to the study conception and design. JRGH and AA conducted the experiments. JRGH analyzed the data and wrote the manuscript. RKB, ETP, and MRA revised the manuscript. All authors read and approved the final manuscript.
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Har, J.R.G., Agee, A., Bennett, R.K. et al. Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon. Appl Microbiol Biotechnol 105, 869–876 (2021). https://doi.org/10.1007/s00253-020-11058-0
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DOI: https://doi.org/10.1007/s00253-020-11058-0