Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Journal Article · 17 June 2018 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.1803745115 · OSTI ID:1454670

Tumen-Velasquez, Melissa; Johnson, Christopher W.; Ahmed, Alaa; Dominick, Graham; Fulk, Emily M.; Khanna, Payal; Lee, Sarah A.; Schmidt, Alicia L.; Linger, Jeffrey G.; Eiteman, Mark A.; Beckham, Gregg T. ; Neidle, Ellen L.

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. We report 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.

• Research Organization: National Renewable Energy Laboratory (NREL), Golden, CO (United States)
• Sponsoring Organization: National Science Foundation (NSF); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
• Grant/Contract Number: AC36-08GO28308
• OSTI ID: 1454670
• Report Number(s): NREL/JA--5100-71408
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 27 Vol. 115; ISSN 0027-8424
• Publisher: National Academy of SciencesCopyright Statement
• Country of Publication: United States
• Language: English