Using the technologies of laboratory-directed evolution and applying the methods
of chemical engineering to devise efficient and robust high-throughput
screens for enzyme evolution offer the promise to revolutionize biological
Input traits could be significantly improved via enzyme engineering. For
instance to improve insect resistance, it may be possible to recombine protective
proteins such as Bacillus thuringiensis
toxin (BT) from multiple independent
sources to create novel variant BT proteins with either increased potency, or
decreased ability to induce resistance in the targeted pest. Alternatively, it may
be possible to improve the efficiency of various pathway enzymes to synthesize
more of a particular protective compound, or changing the chirality of an individual
Output traits present the most easily defined targets for plant improvement.
Plants synthesize a bewildering array of secondary products that have uses
ranging from chemical feedstocks to foodstuffs to pharmaceuticals. By enzyme
engineering, it may be possible to improve the accumulation of desired metabolites.
Plants can efficiently convert CO2
, one of the only natural resources that
continues to become more abundant, into reduced carbon storage compounds
using sunlight as the energy source. It is easy to imagine replacing the enzymes
and pathways used to synthesize storage proteins, carbohydrates, and lipids to
novel pathways to make and store just about any organic molecule we can
conceive. For example, three enzyme pathways for the accumulation of novel
polyhydroxyalcanoates have been successfully engineered into plants (Poirier,
2001). Because plant oils are relatively inexpensive to produce, pathways designed
to produce modified oils with desirable properties as industrial feedstocks
are particularly attractive (Thelen and Ohlrogge, 2002).
Many of the natural enzymes with novel function in pathways such as fatty
acid biosynthesis have been identified. However, alteration of biochemical regulation
of enzyme activity via enzyme engineering of protein stability, sites of
posttranslational modifications, and of allostery represents underexploited
opportunities in plant biotechnology.
Allosteric regulation involves the positive or negative modulation of enzyme
activity after binding of one or more metabolite(s). It represents a particularly
interesting enzyme-engineering target in that the introduction of an enzyme with
altered sensitivity to the interacting metabolite can overcome a potent metabolic
block that cannot be overcome by simply controlling the abundance of the
enzyme. In addition, for many cases, the introduction of an allosterically insensitive variant enzyme should overcome
the metabolic block even in the
presence of the endogenous allosterically sensitive enzyme. Several strategies
can be used to identify enzymes with altered regulation. The first is to identify a
naturally occurring enzyme from a source that does not exhibit allosteric regulation
and to introduce the corresponding gene into the desired host organism. The
second is to perform enzyme engineering and activity screening to identify
variants in which the catalytic activity of the enzyme is maintained, but in
which the binding of the allosteric regulator is disrupted. An excellent example
of overcoming allostery involves starch metabolism. A nonregulated mutant of
the E. coli
ADPG pyrophosphorylase enzyme was identified and introduced into
potato tubers (Ballicora et al.
, 2003), resulting in a 25–60% increase in accumulation
of starch compared to tubers containing the wild-type enzyme (Preiss, 1996). It is
possible that under certain conditions, the metabolic flux into the desired endproduct
may not substantially increase if the allosterically regulated step was
either colimiting or not limiting to the rate of product accumulation. In these cases,
metabolic profiling (Graham et al.
, 2002) can be employed to identify the new
rate-limiting step, and efforts to increase
the activity of this step can be undertaken.
Similar approaches can conceivably be applied to other major forms of
stored carbon such as lipids.
Many aspects of plant architecture, developmental programs, and signal transduction
are regulated by members of families of transcription factors such as
MYBs and MYCs and MAD box proteins. The cauliflower mutant of Arabidopsis
is one of many examples of alteration in expression of a transcription factor
leading to a profound alteration in morphology and development (Kempin et al.
, 1995). One can envisage creating libraries of recombinant chimeras of
transcription factors from these gene families and screening for desired changes
in morphology or development. Such changes might include alterations in the
amount and/or composition of cellulose for improved biomass accumulation.