Physiology and Molecular Biology of Stress ... - KHAM PHA MOI

Metabolic Engineering for Stress Tolerance
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2.4. Network Rigidity and Potential for Negative Alterations of Primary Pathways
Many pathways implicated in stress tolerance are the ones that branch from
primary metabolic pathways. It has been noted that branch points in metabolic pathways
are “rigid” and can not be redirected easily. In certain instances, redirection of
the substrate to a new product has resulted in negative phenotypes because the substrate
is also required by the plant for a primary function. In other instances, even if a
pathway has been installed in a new organism, metabolite accumulation is poor due to
non-availability, poor transport or low concentration or flux of the substrate. Metabolite
accumulation could also be poor due to increased degradation of the reaction
product in the new host. While all the outcomes of a metabolic engineering intervention
cannot be predicted, reiterative engineering steps can be used to overcome network
rigidity problems.
2.5. Identification of Pathways and Genes: The Post-genomic Model
Perception of the stress, signal transduction, activation of transcription factors and
expression of structural genes are the main steps in stress response (Krauss, 2001).
Initially, plants may respond to stress perception by global response, and then by a
more precise or adapted specific stress response (Genoud and Metraux, 1999; Bartels,
2001; Pieterse et al., 2001). Since the availability of whole genome sequence of
Arabidopsis thaliana, identification of genes for use in metabolic engineering for stress
tolerance has enormously improved. Figure 1 shows various steps in the identification,
characterization and use of cloned genes for metabolic engineering for stress tolerance.
Many high throughput techniques such as microarray analyses, proteomics and
metabolomics are available to quickly identify a large number of transcripts, proteins or
metabolites altered in response to stress. While these techniques, as they are applied
most commonly, will not identify a causal relationship between a gene, (enzyme or
metabolite) with stress tolerance, they provide valuable data to make testable hypotheses.
Additional validation can come from mutants affected for their stress tolerance or
correlative data from different genotypes. Availability of T-DNA insertion mutants in
most of the genes in Arabidopsis, makes it possible to test the function of genes whose
functions are currently unknown. Table 2 provides select examples of early rational
metabolic engineering work where a stress tolerant phenotype has been achieved.
While much progress has been made in engineering metabolic and transport traits, the
genes involved in structural and developmental adaptations to stress have not been
understood and used for engineering.