Enzyme Engineering

Enzymes perform the biochemical transformations that direct metabolite flow through metabolic pathways of living cells. Metabolic engineering is made possible via genetic transformation of plants with genes encoding enzymes that selectively divert fixed carbon into desired forms. Genes encoding these enzymes may be identified from natural sources or may be variants of naturally occurring enzymes that have been tailored for specific functionality. The evolution of novel enzyme activities in natural systems provides a context for discussing laboratory-directed enzyme engineering. This process, also called directed evolution, facilitates the expansion of enzyme function beyond the range identified in nature, by altering factors such as substrate specificity, regioselectivity and enantioselectivity. Changes in kinetic parameters such as Kcat, Km and Kcat/Km can also be achieved. Key steps in this process are described, including the selection of starting genes,methods for introducing variability, the choice of a heterologous expression system, ways to identify improved variants, and methods for combining improved variants to achieve the desired activity. Introduction of appropriately engineered proteins into plants has great potential not only for metabolic engineering of desired storage compounds but also for enhancement of productivity by improving resistance to pathogens or abiotic stresses.

Key Words: Enzyme engineering, Directed evolution, Enzyme evolution, Rational design, Sequence space, Variant enzyme, Fitness landscape, Gene shuffling.