|Content of Enzyme Engineering
For ~10,000 years, humans have been tailoring plants to meet their needs. The
vast majority of this crop development occurred as a result of conventional
breeding, that is, by recombining germplasm within the natural breeding
barrier. The results were spectacular improvements in terms of output (harvestable)
traits like yield, and to a lesser extent input (protective) traits such as disease
resistance and stress tolerance. Recently, conventional breeding has been greatly
enhanced by the development of molecular tools. A second wave of improvement
occurred over the past 20 years or so with the development of methods of plant
transformation of genes irrespective of source, with the use of techniques such as Agrobacterium tumefaciens-mediated transformation and DNA particle bombardment.
In contrast to conventional breeding, the major impacts thus far have been
with input traits such as insect and disease resistance. The introduction of engineered
enzymes can be considered as a third wave of plant improvement in which
enzymes with specific tailored properties are introduced into plants with the goal
of conveying specific desired traits. The first example of this was the introduction
of an engineered thioesterase from Garcinia mangostana into canola that resulted in
increased accumulation of stearic acid (Facciotti et al., 1999).
With the emerging wealth of genome information, and the availability of genes
from increasing numbers of organisms, one might ask why engineer genes instead
of simply looking for naturally occurring genes that encode enzymes that already
perform the desired transformation? The simplest answer is that a desired
enzyme might not occur in any natural system. An example might be a biotransformation
for which the substrate is a compound not normally found in nature.
Second, one might identify an enzyme that performs the desired transformation, but does so very poorly. To make the enzyme useful, its activity would need to
be optimized for the desired substrate. Third, the enzyme might have good in vitro
activity, but may behave poorly in the metabolic context of the new host. Thus,
the performance of the enzyme has the potential to be dramatically improved for
use under a specific set of conditions. This could be the case if protein–protein
interactions are necessary for function or if a particular concentration of cofactor
is required. Enzyme engineering can modulate the Km for substrates and cosubstrates.
Finally, the fold of the enzyme may present an inherent limitation to
achieving the optimal catalytic rate for a desired biotransformation, and it might
be better to start with a different protein fold that will allow a higher turnover to
The goal of this chapter is to present the rationale for plant enzyme engineering
in the context of improving plants to meet the increasing and changing demands
of society. To achieve this, I will first lay a conceptual framework for understanding
enzyme evolution as it occurs in nature and then show how the results of
this process may not be ideal for transgenic applications. Next, I will describe
approachesemployed for laboratory evolution of enzymes. Finally, I will summarize
where I see future benefits and applications of these technologies.