Genetically Improved Crops - The Oxford Topic of Food, Politics, and Society - page 78

Likewise, unexpected outcomes are often observed; for example, significant modifica-
tions made to primary Calvin cycle enzymes (fructose-1, 6-bisphosphatase, and phos-
phoribulokinase) have little impact, whereas modifications to minor enzymes (e.g.,
aldase, which catalyzes a reversible reaction) seemingly irrelevant to pathway flux, have
major effects (Hajirezaei et al. 1994; Paul et al. 1995). These observations demonstrate that
caution must be exercised when extrapolating individual enzyme kinetics to the control
of flux in complex metabolic pathways. With evolving “omics” tools, a better understand-
ing of global effects of metabolic engineering on metabolites, enzyme activities, and
fluxes is beginning to be developed. Attempts to modify storage proteins or secondary
metabolic pathways have also been more successful than have alterations of primary and
intermediary metabolism (Della Penna 2006). While offering many opportunities, this
plasticity in metabolism complicates potential routes to the design of new, improved crop
varieties. Regulatory oversight of engineered products has been designed to detect such
unexpected outcomes in biotech crops and, as demonstrated by Chassy et al. (ILSI 2004,
2008), existing analytical and regulatory systems are adequate to address novel metabolic
modifications in nutritionally improved crops (Chassy, this volume).
A number of new approaches are being developed to counter some of the complex
problems in metabolic engineering of pathways. Such approaches include use of RNA
interference to modulate endogenous gene expression or the manipulation of transcrip-
tion factors (Tfs) that control networks of metabolism (Bruce et al. 2000; Butelli et al.
2008; Gonzali et al. 2009; Kinney 1998). Such expression experiments hold promise as
an effective tool for the determination of transcriptional regulatory networks for impor-
tant biochemical pathways. Correctly choreographing the many variables is the factor
that makes metabolic engineering in plants so challenging.
Several new technologies can overcome the limitation of single-gene transfers and facili-
tate the concomitant transfer of multiple components of metabolic pathways. One example is
multiple-transgene direct DNA transfer, which simultaneously introduces all the components
required for the expression of complex recombinant macromolecules into the plant genome.
Nicholson et al. (2005) successfully demonstrated this by delivering four transgenes that rep-
resent the components of a secretory antibody into rice; Carlson et al. (2007) constructed a
minichromosome vector that remains autonomous from the plant's chromosomes and stably
replicates when introduced into maize cells. This work makes it possible to design minichro-
mosomes that carry cassettes of genes, enhancing the ability to engineer plant processes such
as the production of complex biochemicals. Naqvi et al. (2009) demonstrated that gene trans-
fer using minimal cassettes is an efficient and rapid method for the production of transgenic
plants stably expressing several different transgenes. Since no vector backbones are required,
this prevents the integration of potentially recombinogenic sequences, which ensures stability
across generations. They used combinatorial direct DNA transformation to introduce multi-
complex metabolic pathways coding for beta carotene, vitamin C, and folate. They achieved
this by transferring five constructs controlled by different endosperm-specific promoters into
white maize. Different enzyme combinations show distinct metabolic phenotypes, resulting in
169-fold beta carotene increase, six times the amount of vitamin C, and doubling folate produc-
tion, effectively creating a multivitamin maize cultivar (Naqvi et al. 2009). This system has an
added advantage from a commercial perspective in that these methods circumvent problems