To test this idea, Keasling and his students worked with a type of naturally occurring hydrocarbon compounds, called sesquiterpenes, that is widely used in a variety of products. For their model enzyme, they selected a sesquiterpene synthase produced by the Grand fir tree, which has the capacity to develop into any of 52 different sesquiterpenes from a sole substrate.
"This Grand fir sesquiterpene synthase represents the ultimate in promiscuous enzymes," said Yoshikuni. "We were able to take it and construct seven specific and active enzymes synthases. These seven enzymes use different reaction pathways to produce specific products that are as diverse as they can be from one another."
In nature, the divergent evolution of promiscuous enzymes is achieved through trial and error, similar to the way in which the human immune system works. Multiple combinations of many different amino acid substitutions are tested in promiscuous enzymes until an evolutionary path that achieves a desired result is found. The amino acid substitutions that significantly drive molecular evolution are called "plasticity residues."
The Berkeley researchers identified the plasticity residues for the Grand fir sesquiterpene synthase, then systematically recombined mutations of these residues through site-directed mutagenesis, based on a mathematical model developed by Yoshikuni. Construction of the seven sesquiterpene synthases was accomplished with the screening of fewer than 2,500 mutants. An alterative approach, called directed evolution or molecular breeding, that is currently being tested at other laboratories, requires the screening of tens of thousands to a million or more mutants.
"The enzyme synthase was there ready to be evolved, and with our methodology, we were able to rapidly and efficiently evolve it down a pathway of our choice," Keasling said. "We are recapitulating evolution in
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Contact: Lynn Yarris
lcyarris@lbl.gov
510-486-5375
DOE/Lawrence Berkeley National Laboratory
23-Feb-2006