1. UCSF/UCB Joint Graduate Group in Bioengineering,
2. Department of Chemical Engineering, and
3. California Institute for Quantitative Biomedical Research (QB3), University of California at Berkeley, Berkeley, California 94720, USA
4. Synthetic Biology Department, Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94710, USA
5. Department of Pharmaceutical Chemistry and Biopharmaceutical Sciences, University of California at San Francisco, San Francisco, California 94143, USA

Correspondence to: Jay D. Keasling^1,2,3,4 Correspondence and requests for materials should be addressed to J.D.K. (Email: keasling@berkeley.edu).

It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity^{1, 2, 3, 4, 5}, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity)⁶. The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues)^{6, 7, 8, 9, 10}. To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase -humulene synthase^{11, 12}. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.

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