Abstract
Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.
Original language | English |
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Pages (from-to) | 7105-7110 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 115 |
Issue number | 27 |
DOIs | |
State | Published - Jul 3 2018 |
Externally published | Yes |
Funding
We thank David Nelson for assistance in classifying the P450 guaiacol O-demethylases in the CY255A family and Jim C. Spain for discussions of the method. Research was funded by NSF Grants MCB-1361188 and MCB-1615365 (to E.L.N.) and DEB-1556541 (to E.L.N. and M.A.E.). The US Department of Energy (DOE), Energy Efficiency and Renewable Energy, Bioenergy Technologies Office also funded this work via Contract DE-AC36-08GO28308 with the National Renewable Energy Laboratory and its subcontract, XFC-7-70006-01 to the University of Georgia. The US Government retains and the publisher, by accepting the article for publication, acknowledges that the US Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for US Government purposes. ACKNOWLEDGMENTS. We thank David Nelson for assistance in classifying the P450 guaiacol O-demethylases in the CY255A family and Jim C. Spain for discussions of the method. Research was funded by NSF Grants MCB-1361188 and MCB-1615365 (to E.L.N.) and DEB-1556541 (to E.L.N. and M.A.E.). The US Department of Energy (DOE), Energy Efficiency and Renewable Energy, Bio-energy Technologies Office also funded this work via Contract DE-AC36-08GO28308 with the National Renewable Energy Laboratory and its subcontract, XFC-7-70006-01 to the University of Georgia. The US Government retains and the publisher, by accepting the article for publication, acknowledges that the US Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for US Government purposes.
Funders | Funder number |
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US Department of Energy | |
National Science Foundation | MCB-1615365, MCB-1361188, DEB-1556541 |
U.S. Department of Energy | |
Office of Energy Efficiency and Renewable Energy | DE-AC36-08GO28308 |
National Renewable Energy Laboratory | XFC-7-70006-01 |
Small Business Innovative Research and Small Business Technology Transfer | |
University of Georgia |
Keywords
- Acinetobacter
- Evolution
- Gene amplification
- Guaiacol
- P450