Qualitative Control of Metabolites and the Isolation of Desired Biosynthetic Genes

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 functions.

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- metaboliteproducing 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).