For the successful industrial application of secondary metabolite production in
plant cells, both the quantity and quality of metabolites must be improved, that is,
control of the metabolite profile is important. Creation of a new branch to produce
novel compounds by the introduction of a novel gene is a positive approach to
modify this profile. To achieve this end, there are at least two possible approaches.
One is to use biotechnological methods to produce the desired compounds, that is,
heterologous expression of a complete designed biosynthetic pathway in simple
and rapidly growing microorganism systems and/or their use in biotransformation
(Rathbone and Bruce, 2002). The recombinant enzymes would provide novel
catalytic activities, whereas low activity in intact cells often makes such evaluation
difficult. Microbial enzymes have been shown to be useful for the biotransformation
of chemicals. Plant enzymes are much more likely to be biocatalysts, although
they have rather high substrate specificity, for example, a P450, CYP719A1 (Ikezawa et al., 2003).
Another approach is to introduce a new branch pathway into a preexisting
biosynthetic pathway (Sato et al., 2001). A crucial point in creating a new branch in a preexisting pathway is the substrate-affinity/reaction specificity of introduced
enzyme(s). Complete modification of the alkaloid profile with the introduction of C. japonica SOMT cDNA into Eschscholzia californica cells suggested that C. japonica SOMT was superior to E. californica chelanthifoline synthase (Sato et al., 2001); that
is, the alkaloid profile changed from sanguinarine (benzophenanthridine-type) to
columbamine (berberine-type) (Fig. 11.2). However, plant cells do not always
innately show a new pathway; that is, a newly introduced pathway can provide
the substrate for further enzymatic conversion and produce novel compounds
that are not detected in wild-type cells. This result clearly suggests the potential of
plant cells for the production of divergent chemicals after the introduction of a
new branch in a pathway. This experimental result also suggests how plant
cells can obtain a divergent array of metabolites using preexisting pathways.
In either case, it would be very important to isolate genes with novel and desired
Isolation of specific genes
To modify a given pathway, the availability of biosynthetic enzymes is important.
While biosynthetic enzymes with adequate substrate and reaction specificity, or
rational protein engineering, are needed, our knowledge on these subjects is still
limited (Zubieta et al., 2003). Thus, the isolation of specific genes from specialized
cells would be more practical, although it is questionable how we can acquire
good materials to isolate such desired enzymes, cDNAs and genes. While biosynthetic
genes in microorganisms are usually clustered, the genes for secondary
metabolites are not (Hauschild et al., 1998). Previously selected high-
cells would be good candidates for isolating such enzymes and/or
genes (Hashimoto and Yamada, 2003). For example, berberine-producing C. japonica cells have been shown to be very useful for isolating biosynthetic
genes, as seen in oil gland cells (Lange et al., 2000; Morishige et al., 2002). Similarly,
the comparison of morphine producing and nonproducing Papaver spp. has been
used to identify the gene(s) specific for alkaloid production (Ziegler et al., 2005). In
either case, the further characterization of ESTs by microarray or Northern analysis
should be effective for evaluating the functional linkages of genes. Another
possibility is the use of compounds to induce metabolism. The cDNA-amplified
fragment length polymorphism (AFLP) method is an attractive alternative for
identifying genes involved in plant secondary metabolism, in combination with
targeted metabolite analysis (Goossens et al., 2003b). Alternatively, if we could
isolate a general transcription factor(s) that targets secondary metabolism, the use
of transgenic cells as the starting material could become possible. One example is
provided by Arabidopsis thaliana overexpressing the PAP1 gene encoding an MYB
transcriptional factor. This line was used to identify a novel gene involved in
flavonoid biosynthesis using the integration of metabolomics and transcriptomics
(Tohge et al., 2005). A proteomics approach can also be useful for examining
proteins by two-dimensional gel electrophoresis and internal peptide microsequencing.
Using this approach, a representative enzyme in morphine biosynthesis
could be detected within the serum fraction of latex of opium poppy (Decker et al., 2000). As shown above, a recent trend is to avoid biochemical purification and to
directly isolate candidate clones by a combination of expression pattern analysis
and homology-based screening (Hashimoto and Yamada, 2003). However, our
understanding of the resulting data is limited due to a fundamental lack of
biochemical and physiological knowledge about network organization in plants,
although the development of metabolomic methods and tools is progressing
rapidly (Tohge et al., 2005; Weckwerth and Fiehn, 2002).
Quality Control of Metabolites
The simple composition of metabolites is desirable for industrial production.
The simplest approach to producing desired compounds is to reconstruct entire
biosynthetic processes In vitro, as mentioned above. So far, a considerable number
of genes involved in alkaloid biosynthesis have been cloned and expressed in
E. coli or insect cells (De Luca and Laflamme, 2001; Facchini, 2001; Hashimoto and
Yamada, 2003; Rathbone and Bruce, 2002; Verpoorte and Memelink, 2002). While
microbial cells have less capacity to store metabolites, heterologous systems could
be useful for bioconversion.
A more promising approach is the downregulation of gene expression for an
undesired pathway. Downregulation using antisense material and cosuppression
have been shown to be effective since mint plants transformed with the antisense
version of menthofuran synthase cDNA produced less than half of the undesired
monoterpene oil component than did wild type (Mahmoud and Croteau, 2001).
Suppression of a P450 hydroxylase gene in plant trichome glands has also been
associated with the accumulation of cembratriene-ol and enhanced resistance
against aphids (Wang et al., 2001).
The recent development of the RNA interference (RNAi) method using
double-stranded (ds)RNA-induced posttranscriptional and/or transcriptional
silencing is a more efficient method for modifying a pathway to shut down
(Wang and Waterhouse, 2002; Waterhouse and Helliwell, 2003; Wesley et al., 2001).
While transgenic E. californica cells with antisense BBE RNA lost alkaloid productivity
with no accumulation of intermediate reticuline (Park and Facchini, 2000;
Park et al., 2002), our experiment with the dsRNA expression vector for BBE
(BBEir) evidently induced reticuline accumulation with the reduction of endproducts
(Fujii et al., 2007). This result clearly shows that this new tool is useful
for metabolic engineering and that plant cells are capable of storing intermediates
even when the metabolic pathway has been altered. A note of caution about the
selection of target sequences must be included because short stretches of oligonucleotides
that perfectly match a gene sequence may effectively lead to silencing
of homologous genes (Ishihara et al., 2005). However, the silencing of undesired
genes and the elimination of certain proteins can also provide a way to alter
metabolic pathways. Accumulation of reticuline in transgenic opium poppy
with a COR RNAi vector could lead to silencing effects, while disruption of
enzyme complex for morphine alkaloid biosynthesis after reticuline also might
be involved in the accumulation of reticuline (Allen et al., 2004).