|Content of 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