Functional genomic approaches for engineering of secondary metabolic pathways of medicinal and aromatic plants
Authors: Dr. Vinay Kumar and Dr. Y.M.Shukla ^1
1Department of Biochemistry, Anand Agricultural University, Anand (Gujarat).

Introduction

In India, about 7500 species of higher plants are known for their medicinal uses (Shiva, 1996) which are supposed to be the highest in the whole world. Ayurveda, the oldest medical system in the Indian subcontinent, has alone described usages of approximately 2000 medicinal plant species, followed by the Siddha and Unani. Nearly 80% of African and Asian population depends on traditional medicines for their primary healthcare. In India, about 80% of the rural population uses medicinal herbs or indigenous systems of medicine(ISM). It is estimated that about 25% of the drugs prescribed worldwide are derived from plants, and many others are synthetic analogues built on prototype compounds isolated from plant species in modern pharmacopoeia (Kala and Sajwan, 2007). The total of about 252 drugs listed in World Health Organizations (WHO's) essential medicine list of which 11% is exclusively of plant origin.

What are secondary metabolites?

Secondary metabolites, also referred to as natural products, are the products of metabolism, which are not essential for normal growth, development or reproduction of an organism. These compounds serve to meet the secondary requirements of the producing organisms. They empower them to survive inter-species competition, provide defensive mechanisms and facilitate reproductive processes. Many secondary metabolites have proved invaluable as antibacterial or antifungal agents, anticancer drugs, and cholesterol lowering agents, immuno-suppressants, antiparasitic agents, herbicides, diagnostics, and tools for research (Vaishnav and Demain, 2010). At least 1,00,000 such secondary metabolites are now known to occur in 50,000 plant species and ~4,000 new secondary metabolites are being discovered every year from a variety of plant species (Galera et al., 2007). Secondary metabolites are characterized by enormous chemical diversity and every plant has its own characteristic set of secondary metabolites. Based on their biosynthetic origins, plant secondary metabolites can be structurally divided into three major groups, viz., terpenes or isoprenoids, nitrogen containing secondary metabolites and phenolic compounds. The occurrence of secondary metabolites is the plant kingdom and may be restricted to a particular taxonomic group (genus, species and family). They may be synthesized in specialized cells, tissues and organs even at a specific developmental stage of the plant (Caldentey and Inze, 2004).

Approaches for Engineering of secondary metabolic pathways:

Engineering of secondary metabolic pathways in plants requires a thorough knowledge of the whole biosynthetic pathway and a detailed understanding of the regulatory mechanisms controlling the onset and the flux of the pathways. The following strategies could be used to enhance or modify the production of desired plant metabolite (Caldentey and Inze, 2004)
(1) Decrease the catabolism of the desired compound;
(2) Enhance the expression or activity of a rate-limiting enzyme;
(3) Prevent feedback inhibition of a key enzyme;
(4) Decrease the flux through competitive pathways;
(5) Enhance expression or activity of all genes involved in the pathway;
(6) Compartmentalization of the desired compound; and
(7) Conversion of an existing product into a new product.

Functional genomic approaches for engineering of secondary metabolic pathways:
Genetic engineering of a secondary metabolic pathway aims to either increase or decrease the quantity of a desired compound or group of compounds and/or production of novel compounds:

1. Upregulation or over expression of the gene(s)/enzyme(s)
(i)Single gene engineering;
(ii) Multiple gene(s) engineering (simultaneously or sequentially) and;
(iii) Engineering of regulatory genes

2. Down regulation or functional knockout of the gene(s)/enzyme(s)
(i) Antisense RNA technology;
(ii) Co-suppression and;
(iii) RNA interference (RNAi)

Up regulation or over expression:
Single gene engineering: Single gene manipulations of rate limiting enzyme of the biosynthetic pathway may leads to the increase in concentration of desired metabolite. This approach can also be used to extend a metabolic pathway in a heterologous plant. Yun et al., (1992) introduced the cDNA of gene encoding hyoscyamine-6-hydroxylase H6H from Hyoscymus niger into Atropa belladonna resulted in the production of scopolamine in A. belladonna, where the tropane alkaloid pathway stops at L -hyoscyamine.

Multiple gene engineering: To overcome the limitations of the single- gene approach the alternate way to simultaneously transform plants with genes encoding enzymes that act at different steps of a biosynthetic pathway. It has become increasingly common for multiple genes to be introduced into plants either stepwise (by crossing independent single gene transgenics) or by simultaneous multiple gene transfer using particle bambadment, or cocultivation (Sato et al., 2001).

Engineering of regulatory genes: The use of regulatory genes can be an alternative to overcome the limitation of the structural gene approach for manipulation of entire or part of metabolic pathways. Transcription factors (TFs) act as master regulators of complex pathways, two TFs genes octadecanoid -responsive Catharanthus AP2 domain proteins (ORCA2 and ORCA3) were cloned and known to regulate a number of genes in primary as well as secondary metabolism leading to the production of terpenoid indole alkaloid. (Gantet and Memelink, 2002).

Down regulation or functional knockout:

To decrease the production of a certain unwanted (group of) compound(s) several approaches are possible. An enzymatic step in the pathway can be knocked out by reducing the level of the corresponding mRNA or protein.

Antisense RNA technology: Antisense RNA has the opposite sense to mRNA. The presence of complementary sense and antisense RNA molecules in the same cell can lead to the formation of a stable duplex, which may interfere with gene expression at the level of transcription, RNA processing or possibly translation.

Cosuppression: refers to the ability of a sense transgene to suppress the expression of a homologous endogenous gene (Napoli et al., 1990). Use of antisense RNA and cosupression approaches sometimes may fail to block the activity of an enzyme encoded by multigenes. RNAi technology provides an alternative to block the activity of such enzymes that are not only encoded each by a multigene family but are also expressed across a number of tissues and developmental stages (Borgio, 2009). RNA interference (RNAi) is a double-stranded RNA (dsRNA) induced gene-silencing phenomenon, conserved among various organisms, including animals and plants (Fire et al., 1998). The effect of the silencing of codeinone reductase gene (COR) in the opium poppy (Papaver somniferum), using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi) was studied and analysis of transgenic line showed that after gene silencing, the precursor alkaloid (S)-reticuline-seven enzymatic steps upstream of codeinone accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine specific branch. This study showed that the gene silencing in transgenic opium poppy and metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline (Allen et al., 2004).

Conclusion:
• Over expressing multiple key-enzyme genes in the target bioengineering pathway is a promising way to manipulate the biosynthetic pathways.
• RNAi technology has potential to block the activity of enzymes that are not only encoded by a multigene family but are also expressed across a number of tissues and developmental stages. Knockdown the activity of codeinone reductase genes through RNAi in transgenic opium poppy which, resulted in accumulation of nonnorcotic compound (s)-Reticuline.
• Extensive studies are required to elucidate the secondary metabolic pathways and the genes involved in these pathways at least in the major medicinal and aromatic plant species.
• High throughput screening systems could be applied for discovery of new functional compounds/ phytochemicals of medicinal importance.
• Identification and deployment of cis and trans acting factors that regulate the temporal and spatial gene expression of secondary pathways.

References
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About Author / Additional Info:
I am Biotechnology professional with Ph.D in Plant Molecular Biology and Biotechnology and working as Scientist, Biotechnology