EASy Evolution Accelerates Enzyme Engineering

Microbes evolve new traits quickly, but not quickly enough for scientists eager to direct the evolution of microbes toward desirable ends. Usually, scientists must wait for chance occurrences to create an evolution-accelerating change called gene amplification, which multiplies the copies of a gene that may acquire new and potentially beneficial mutations. To make gene amplification fast and easy, scientists based at the Department of Energy (DOE) and the University of Georgia developed a method called, appropriately enough, EASy.

EASy stands for Evolution by Amplification and Synthetic Biology. It enables the back-to-back incorporation of hundreds of copies of a gene into a cell. This region of repetitive DNA provides the cell with a means to undergo accelerated evolution of this gene. If this gene codes for an enzyme of interest, EASy can be used to expedite the generation of better-performing enzymes.

“We can make many, many random changes and identify those that are of interest using evolution,” said Christopher Johnson, Ph.D., a molecular biologist at the DOE’s National Renewable Energy Laboratory (NREL) who participated in the development of EASy. Along with his colleagues, Johnson used EASy to dramatically improve an enzyme’s ability to break down biomass.

Details of the work appeared June 18 in the Proceedings of the National Academy of Sciences, in an article entitled “Accelerating Pathway Evolution by Increasing the Gene Dosage of Chromosomal Segments.” This paper describes how EASy was used to create tandem arrays of specific DNA segments in a bacterium, Acinetobacter baylyi. By exploiting these tandem arrays, the scientists expedited the bacterium’s ability to explore variants of a gene introduced from another bacterium, Amycolatopsis. Ultimately, an unusual fusion of enzymes from the two species of bacteria was achieved.

“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,” wrote the article’s authors. “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 ∼۱,۰۰۰ 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).”

Essentially, the researchers inserted DNA that encodes the enzyme GcoA from the bacteria Amycolatopsis into A. baylyi, placing it adjacent to the gene that encodes the CatA enzyme. The EASy technique resulted in the unusual fusion of two genes into a single gene encoding a chimeric enzyme.

The trait afforded by this chimeric enzyme was the ability to more efficiently convert a component of lignin—a particularly resilient part of plant biomass—into fuels, and a precursor of plastics such as nylon lignin comprises about 30% of biomass.

“It’s a matter of conversion efficiency,” said Jeffrey Linger, Ph.D., an NREL research and co-author of the PNAS paper. “If you’re not using that 30%, you’re throwing it away. We’re trying to capture that 30%.”

Reference

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