Creating plant molecular factories for industrial and nutritional isoprenoid production
Graphical abstract
Introduction
Isoprenoids, also known as terpenoids represent the largest and oldest class of natural products documented, consisting of >40 000 different molecules [1]. Biosynthetically all isoprenoids are related via a common five carbon building block (isopentenyl pyrophosphate; IPP). Commercially isoprenoids are used in cosmetics and fragrances, and as colourants and nutritional supplements in foods and feeds. Additionally, numerous isoprenoids have important medicinal properties, such as the anti-cancer agents Taxol and the isoprenoid-derived indole alkaloid vincristine and vinblastine [2]. Their industrial relevance also means they are compounds of high value with global markets in the range of $1 billion per annum (www.researchandmarkets.com). A contributing factor to the high cost of these molecules resides in the fact that they are mainly produced in low yields by slow growing plant species that are not readily amenable to agricultural production. It is therefore not surprising that total or semi-synthetic chemical synthesis is presently the method of choice for obtaining many of these isoprenoid molecules. However, their structural complexity makes chemical synthesis expensive, difficult and prone to the formation of non-biological stereoisomers and contamination with reaction intermediates. There is also a significant environmental impact as the precursors used are typically derived from the chemical refining of fossil fuels [3].
In addition to their value as speciality or bulk chemicals, isoprenoids such as carotenoids (provitamin A) and tocopherols (vitamin E) are essential components of the human diet. Moreover, nutritionally enhanced foods are advocated by most national governments [4]. Therefore, enhancement in crop plants such as rice, maize and tomato to confer improved nutritional and consumer quality traits is an area of intense research and development.
The formation of isoprenoids is a complex process in plants, that is compartmentalised and under developmental regulation. The biosynthetic pathway(s) can be divided into first, the formation of the IPP; second, the formation of prenyl phosphates and third, biosynthesis of isoprenoid subgroups. Since the discovery of the mevalonic acid (MVA) pathway in the 1950s, it was assumed that IPP was synthesised from acetyl-CoA via MVA. However, experimental data consistently indicated that the MVA pathway could not account exclusively for the formation of plastid-derived isoprenoids. In the early 1990s, retro-biosynthetic labelling conclusively established the presence of an alternative MVA-independent pathway for the formation of IPP and dimethylallyl pyrophosphate (DMAPP), termed the methyl d-erythritol phosphate pathway (MEP) [5].
Prenyl phosphates represent the central backbone of the isoprenoid pathway from which all isoprenoid sub-groups are derived. The five carbon substrates used are IPP and DMAPP. These molecules lead to the formation of longer chain prenyl lipids such as geranyl pyrophosphate (GPP, C10), farnesyl pyrophosphate (FPP, C15), geranylgeranyl pyrophosphate (GGPP, C20), octaprenyl pyrophosphate (C40) and solanesyl pyrophosphate (C45). GPP, FPP and GGPP are the main products synthesised in plants. The presence and action of GPP, FPP and GGPP synthases is key to the prenyl lipids produced and thus the formation of independent branches of isoprenoid biosynthesis that utilise these prenyl precursors (Figure 1). A vast array of isoprenoid subgroups exist, these include monoterpenes (C10 derived) such as geraniol, sesquiterpenes (C15 derived), which includes artemisinin, diterpenes (C20 derived) such as taxanes, triterpenes (C30 derived), for example, phytosterols and carotenoids (C40 derived) such as β-carotene.
Following the elucidation of rudimental knowledge of isoprenoid formation, the last decades have seen an explosion in our ability to implement genetic intervention (metabolic engineering and Marker Assisted Selection, MAS) to alter isoprenoid levels in plants. The following sections will provide notable examples of rational strategies that have been used to achieve successful production of certain isoprenoids or which provide exploitable fundamental knowledge that could lead to future advances. An attempt will be made to judge the levels of socio-economic impetus required to fully adopt these new biosources as foods and production platforms.
Section snippets
Strategies used for the metabolic engineering of isoprenoid production in plants
Metabolic engineering strategies are summarised in Figure 2, for convenience we have used the plastid compartment to illustrate the approaches used. Initial efforts focused on the modulation of single key enzymatic steps in a pathway. These enzymes typically had the highest flux coefficient among the pathways components and thus, their modulation increased metabolic flux through the targeted pathway. Alternatively, the first committed step in a pathway was targeted in order to divert precursors
The future exploitation of isoprenoid formation in plants
The present molecular resources and tools now available have enabled the development of excellent new prototypes for the production of useful isoprenoids. However, the field is on the horizon of new paradigm changing opportunities for crop improvement using targeted genome engineering [43]. Particularly, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system [44] which is emerging as a transformative tool, due to its ease of engineering,
Conclusions and perspectives
This review highlights the plethora of strategies and emerging technologies available for the production and enhancement of nutritional and industrially useful isoprenoids in plants. It can be concluded that, in most cases, it is now possible to rapidly progress through proof of concept studies and deliver genotypes with high titres of valuable isoprenoids or enhanced levels of nutritional related isoprenoids, for direct nutritional intervention. So the question remains; why these notable
Conflict of interest
The authors wish to confirm that there are no known conflicts of interest associated with this publication.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
The authors acknowledge the EUFP7 DISCO No. 613513 and TomGEM H2020 programme No. 679766 for support. We apologise to the many authors whose papers we could not cite or overlooked here due to space limitations.
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- a
Joint first authorship and these authors contributed equally.