In this article we will discuss about the metabolic engineering of monoterpene.
Terpenoids are otherwise known as isoprenoids are the largest family of natural products. They are well known for their constituents of the essential oils. Monoterpenes are commercially significant because of their extensive use in food, cosmetic and pharmaceutical industries. The terpenoids are made up of few to thousands of isoprene units.
Biosynthesis of terpenoids takes place by the condensation of dimethyallyl diphosphate (DMAPP) and isopentanyl phosphate (IPP). The sequential head to tail addition of IPP units to DMAPP produce geranyl diphosphate (GPP) by GPP synthase, farnesyl diphosphate (FPP) by FPP synthase and geranyl diphosphate (GGPP) by GGPP synthase.
Monoterpenes are C10 class of isoprenoids, comprises nearly 1000 colourless volatile and liphophilic metabolites. Monoterpenes are produced exclusively from GPP which is often cyclized to produce the various monoterpene sub families. The biosynthesis of terpenoids in plants is regulated at the subcellular level.
The primary carbon skeleton of monoterpenes and diterpenes are produced in plastids and the backbones of sesquiterpenes and diterpenes are manufactured in the cytoplasm. Further enzymatic modifications to these backbones also occur in various cell compartments like endoplasmic reticulum, where terpene back bones are hydroxylated. Mitochondria also participate in isoprenoid biosynthesis.
In transgenic applications availability of excess of precursors can result in increased production of terpenes. Therefore, in one of the strategy, over production of GPP synthase and down regulation of the production of GPP synthase is expected to produce excess of GPP and consequently accumulate monoterpene end products.
One of the most committed enzymes in monoterpene biosynthesis is monoterpene synthase or cyclases, which converts GPP (universal precursor) into various monoterpene families. This enzyme is an attractive target for the engineering of monoterpene metabolism.
Manipulating the expression of genes for monoterpene synthase could increase yield of the targeted transgenic plants. Expression of limonene synthase in peppermint and cornmint has been found to increase yield and alter monoterpene composition in these two species.
One of the most striking finding is that sesquiterpene production is significantly enhanced if the corresponding sesquiterpenes synthase is targeted to the plastids and combined with a plastidic Farnesyl diphosphate synthase (FPS).
For example, production of amorpha-4, 11-diene, and the precursor of the antimalarial drug artemisinin could be improved to more than 40,000 fold by plastid targeting to the appropriate terpene synthase together with FPS, instead of using native FPS activity and cytosolic targeting.
Although mono-terpene synthase required for limonene production is generally plastid region, cytosolic expression of limonene synthase with geranyl diphosphate synthase (GPS) boosted production of the flavor compartment.
However, limonene production was higher when the synthase and GPS were co-expressed in plastids. This accomplishment in engineering plastid sesquiterpene production confirms the potential of this compartment (Fig. 17.8A) to supply large amounts of precursors for (mono) terpene production.
DMADP – dimethylallyl diphosphate
FPS – FDP synthase
FDP – farnesyl diphosphate
GPS – GDP synthase
GDP – geranyl diphosphate
HMGR – HMG-CoA reductase
IDP – Isopentanyl diphosphate
MTS – Monoterpene synthase
MVA – Mevalonic acid
STS – Sesquiterpene synthase
MEP – Methyl erythritol phosphate
CYTP 450 – Cytochrome P450 hydroxylase.
Another classic example of metabolic engineering of monoterpene is the production of alternation of peppermint oil and has been used as model system for the Study of monoterpene biosynthesis. The essential oil of peppermint consists mainly of p-mentbane monoterpene with menthol as chief component. These essential oil components are affected severly by high temperature and low light intensities.
These adverse environmental conditions favour the production and accumulation of pulegone and menthofuran, both of which are responsible for bad odours. Metabolic engineering is adopted to minimise the production of undesirable metabolites peppermint and also maintain the maximum production of menthol.
In transgenic work reducing the expression of cytochrome P450 menthofuran synthase (MFS) gene, which is responsible for the conversion of pulogone to menthofuran using antisense strategy resulted in considerable (nearly 75%) decrease in the levels of menthofuran in peppermint oil and even virtual elimination of the menthofuran from the oil. These results reflected that it is possible to modify monoterpene biosynthetic pathway through metabolic engineering.
Another attractive candidate for monoterpene engineering is the conifers, which are used for pulp and timber production. These economically important trees produce terpene based oleoresins to combat insect attack. Oleoresin is a mixture of mono, di and sesquiterpene. Thus, insect resistant transgenic trees can be produced by manipulating the composition of oleoresin, which can deter the insects and also disrupt insect aggregation.