Some example includes amino acids, fatty acids, and alcohols in bacteria; ... Plant metabolic engineering has been attempted in all these directions and promising ... Gibberellin, abscisic acid and brassinosteroid phytohormones, phytosterols and .... Negative feedback regulation of enzymes can result from product inhibition ...
Chapter 1 Introduction 1.1 Metabolic Engineering Metabolic engineering is a 25 years young branch in the field of genetic engineering (G. N. Stephanopoulos, Aristidou, and Nielsen 1998). It has been defined as the “directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or the introduction of new genes with the use of recombinant DNA technology” (G. Stephanopoulos and Sinskey 1993). Basically, the goal is to construct cell systems that can produce large amount of fuels, chemicals, and pharmaceuticals in a cost effective manner (Kern et al., 2007; Yang et al., 2010). To achieve this target, metabolic engineering relies on knowledge of cell physiology and genetic engineering at its core. These two basic elements form indispensable components of metabolic engineering. The field is further strengthened with incorporation of expertise from various fields like bioinformatics, chemical engineering, bioreactor technology etc. In the past few years, metabolic engineering has undergone radical advancement due to two major factors (1) Ability to chemically synthesize DNA at low cost and (2) Development of high throughput omics technology (Woolston, Edgar, and Stephanopoulos 2013). Implementing “omics” technology to different types of biological systems (bacteria, yeast, fungi, plant) has generated large amount of gene, protein and metabolite data (Pickens, Tang, and Chooi 2011). Due to these developments, the capabilities of metabolic engineering in various organisms have increased tremendously. It now has ability to manipulate large number of biochemical reactions in wide variety of biological systems including model as well as non model organisms. The endeavor in field of metabolic engineering has given encouraging results. A comprehensive overview of number of products spanning several industries for which metabolic engineering has been successfully applied to manipulate product yield up to date is shown in Figure 1.1. These
industrially important products usually comprise of primary and secondary metabolites produced by various living organisms ranging from prokaryotes like bacteria to eukaryotes like yeast fungi, plant etc. Some example includes amino acids, fatty acids, and alcohols in bacteria; alcohols in yeast; organic acids, vitamins, extracellular enzymes, antibiotics in fungi; phenolics, terpenoids, alkaloids in plants (Fisher et al., 2014). Amongst these biological systems, metabolic engineering has been extremely successful in manipulating bacteria, fungi and yeast to produce metabolites at industrially desired levels. Many of these products are available in market and have been commercially successful (Erickson et al., 2012). Unfortunately, in case of plants metabolic engineering attempts have not been able to repeat success story. Decades of research work in plant metabolic engineering has not fulfilled expectations of industrial scale productivities for plant secondary metabolites. Metabolic engineering has immense potential to dramatically enhance cell productivity as proven in case of bacteria, yeast and fungi and therefore, such opportunity cannot be let go off in case of plants. Driven by this motivation, researchers pursue this field ever the more in plants facing all challenges it has to offer so as to meet desired goal of cost effective industrial level production.
Figure 1.1: Metabolic engineering of various industrially valuable products in different living organisms like bacteria, yeast, fungi, plant. Amino acids, organic acids, fatty acids and alcohols are primary metabolites. Terpenoids, alkaloids and phenolics are secondary metabolites
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1.2 Plant Metabolic Engineering Plants, in general, are subject of extreme interest for metabolic engineering. Plants have potential to serve as cost effective sustainable production systems for industry (Yuan and Grotewold 2015). The whole idea of metabolic engineering is to increase metabolic output of plant. Plant metabolic engineering has potential to generate endless opportunities in the field of environment, agriculture, medicine, energy etc. Key applications of plant metabolic engineering include enhancing nutrient content of crop plants; introducing self nitrogen fixation capacity in plants, enhancing production of industrially valuable secondary metabolites in plants, enhancing bio-fuel production from plant and increase photosynthetic efficiency as shown in Figure 1.2 (Lau et al., 2014). Plant metabolic engineering has been attempted in all these directions and promising results have been obtained. However, more research is needed to exploit full potential of plant metabolic engineering. Amongst these applications, most lucrative field of action is metabolic engineering of plant secondary metabolites.
Biofuel
Self nitrogen fixation
Plant metabolic engineering
Enhance photosynthetic efficiency
Alter primary/ secondary metabolite content Figure 1.2: Plant metabolic engineering: Key areas of interest
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1.3 Metabolic Engineering of Plant Secondary Metabolites About 200,000 different secondary metabolites are estimated to occur in plant kingdom (Staniek et al., 2013). Almost 20–30% of pharmaceutical drugs in market are derived from secondary metabolites obtained from plants (Raskin et al., 2002). Many of these valuable secondary metabolites are obtained from medicinal plants which grow slowly and are difficult to cultivate. For example, 10,000 kg of Pacific yew bark produced less than 1kg of anti-cancer compound paclitaxel (Taxol). Therefore, large scale production requires number of plant or trees to be destroyed leading to environmental pressure. Chemical synthesis could be an alternative approach to produce these chemicals but is often impractical and uneconomical due to their complex structures. Conventional breeding is also an active area of research but is a slow process. In such a scenario metabolic engineering of plants could emerge as an effective solution to dramatically enhance production of secondary metabolites at industrially desired levels.
1.3.1 Secondary metabolites In general, secondary metabolites are not required for immediate survival of cell but they have many key role to play in cell defence. Functional role of secondary metabolites in plant ranges from the ecology to defence. Secondary metabolites are involved in protecting plant against both biotic and abiotic stresses, ecological roles such as attractants for pollination and/or repellents for phytophagy (Irito and Faoro 2009). They also provide colour and scents to plant reproductive organs etc. Significance of plant secondary metabolites extends into human realm as well. These compounds exhibit many biological activities of pharmacological significance like anti cancer, antibiotic, antihypertensive, antidiabetic, hepatoprotective etc. In addition these compounds are also used as food colours, dyes, perfumes etc. All these compounds arise from a rather limited number of chemical scaffolds divided into three major groups: phenolic, terpenoids and alkaloids
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Phenolics Phenolics, are a class of chemical compounds
consisting of a hydroxyl group (—OH)
bonded directly to an aromatic hydrocarbon group. Phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule. About 8,000 phenolic compounds, also called phenylpropanoids are reported in plants (Staniek et al., 2013). Phenylpropanoid diversity consists of hydroxycinnamic acids, cinnamaldehydes, monolignols, coumarins, flavonoids, phenylpropenes, stilbenes, lignanas, lignins, suberiins etc (Vogt 2010). Some important phenolics include vanillin used in food industry, curcumin, reservatrol in pharmaceutical industry. Terpenoids Terpenes are hydrocarbons resulting from the combination of several isoprene units. The isoprene unit has the formula CH2=C(CH3)CH=CH2. Terpenoids are modified terpenes, wherein methyl groups have been moved or removed, or oxygen atoms added. Terpenoids also known as isoprenoids represent chemically and functionally most diversified class of primary as well as secondary metabolites. Primarily About 40,000 are expected to occur in plants (Tholl 2006). Gibberellin, abscisic acid and brassinosteroid phytohormones, phytosterols and carotenoid pigments are primary metabolic terpenoids which form a portion of terpenoids in plants. However, the majority of plant terpenoids are secondary metabolites. Important secondary metabolite terpenoids include menthol used in flavor and fragrance industry; abietic acid, a diterpenoid used in lacquers, varnishes and soap, natural rubber used as biological material in production of heavy-duty tires, latex products, etc (Tholl 2006) Alkaloids Alkaloids are compounds which derive from amino acid and a heterocyclic ring with nitrogen. The nitrogen in the alkaloid molecule is derived from amino acid metabolism. Since the amino acid skeleton is often largely retained in the alkaloid structure, alkaloids originating from the same amino acid show similar structural features. More than 21,000 alkaloid structures are currently known (Farré et al., 2014). A majority of alkaloids are amino acid derivatives grouped
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on the basis of their precursor and chemical structure (Iriti and Faoro 2009). Accordingly, pyrrole alkaloids arise from leucine; pyrrolidine, tropane and pyrrolizidine alkaloids from ornithine; piperidine, quinolizidine and indolizidine alkaloids derive from lysine; catecholamines, isoquinoline, tetrahydroisoquinoline and benzyltetrahydroisoquinoline alkaloids originate from tyrosine; indolamines, indole, carboline, quinoline, pyrrolindole and ergot alkaloids come from tryptophan and imidazole alkaloids from histidine. Anthranilic acid is the precursor of quinazoline, quinoline and acridine alkaloids (O’Connor and Maresh 2006). Important alkaloids include morphine, codeine, vincristine, vinblastine, atropine, cocaine, quinine, taxol, camptothecin etc for pharmaceutical industry. The enormous diversity is based on substrate and/or regiospecific enzymes that evolve the molecular backbones through different chemical modifications e.g., hydroxylation, methylation, acylation and glucosylation (Dixon 2001; Vogt 2010). Such series of enzyme-catalyzed chemical reactions leading to production of a particular metabolite is classically defined as metabolic pathway. Many such secondary metabolic pathways exist in plants such as phenylpropanoid pathway, terpenoid pathway, alkaloid pathway etc and have been studied in detail with reference to plants, to understand origin, functionality and mechanism of action of these metabolites. 1.3.1.1
Linear secondary metabolic pathway: The fundamental concept
Although debatable, the basic definition of metabolic pathway is a series of enzyme-catalyzed chemical reactions occurring within an organism (Caspi, Dreher, and Karp 2013). Decades of research has resulted into core metabolic pathways including primary metabolic pathways as well as early secondary metabolic pathways being well characterized. Secondary metabolite pathway arises from primary metabolites as shown in Figure 1.3. Phenylpropanoid pathway has its origin from amino acid phenylalanine and this pathway then gives rise to diverse phenolic compounds. Phenylpropanoid pathway branches out to give rise to flavonoid pathway producing flavonoids, anthocyanin pathway producing anthocyanin, and lignin pathway producing lignin. Terpenoid pathway arises from Isopentenyl pyrophosphate (IPP) five carbon precursor. Diverse alkaloids do not have a common origin point and arise from different amino acids. In this context, some preliminary information has also been generated at
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enzyme and gene levels for biosynthetic pathways of phenylpropanoid including flavonoid, lignin, anthocyanin, terpenoids and alkaloids (Farré et al., 2014). Many structural genes coding for enzymes involved in few steps in secondary metabolite pathway have been cloned and gene sequences are available in databases like NCBI. However, various genes coding for entire pathways have yet to be decoded (Farré et al., 2014).
C02 +H20 Photosynthesis Primary metabolism
Alkaloid pathway(s)
Phenylpropanoid pathway
Terpenoid pathway
(Common origin)
(Different origins)
(Common origin)
Sesquiterpenes Monoterpenes
Alkaloids
Phenolics Lignins
Diterpenes
Terpenes
Sesteterpenoids Flavonols
Polyterpenes
Anthocyanin Secondary Metabolism Figure 1.3: Brief overview of various metabolic pathways and their branching
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1.3.1.2
Secondary metabolic pathway: The evolved concept
With time, it has been proved that metabolic pathway is not a simple series of linear reactions but actually a more complex network regulated at many levels (Kooke and Keurentjes 2012). Each and every step of secondary metabolic pathway like expression of genes, activity of proteins, concentrations and accumulation of intermediates and products etc involved in normal functioning of secondary metabolite pathway is fine tuned by many regulatory elements. These regulatory elements form an intricate net where each string is affected by the other. These regulatory elements are present within the plant as well as outside the plant system and directly or indirectly govern expression of secondary metabolite pathway. These multi-dimensional regulatory inputs are discussed as follows: A) Molecular regulation: It operates at hierarchical levels. It originates at the level of transcription moving on to translation and enzyme activity or usually a combination of them (Vemuri and Aristidou 2005). For eg. (i)Transcriptional regulation: Transcriptional factors (TFs) activate are DNA sequences which bind to promoter region of gene coding for biosynthesis pathway and activate or repress expression of secondary metabolite biosynthetic genes in response to developmental and/or other environmental cues. Some TFs many not bind directly but act indirectly. For example, most of the structural genes in the anthocyanin biosynthesis pathway are coordinately regulated by transcription factors (TFs), namely R2R3 MYB, basic helix–loop–helix (bHLH) and WD-repeat (WDR) proteins. (Patra et al., 2013) (ii) Translational regulation: It can occur during or after translation via RNA protein complexes or proteolysis. For example, 26S ubiquitin proteasomal system (UPS) is control machinery which can regulate anthocyanin biosynthetisis through proteolysis of important TFs. UPS also regulates the turnover of metabolic enzymes of phenylpropanoid pathway (Zhang et al., 2013) (iii) Enzyme activity regulation: At enzyme level, almost all metabolic pathways are subject to feedback regulation by which the product influences its own production often by controlling activities of pathway enzymes (Tzin and Galili 2010). Negative feedback regulation of enzymes can result from product inhibition or allosteric
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inhibition, in which the regulatory molecule interacts at an allosteric site other than the active site to inhibit the enzyme activity. (B) Temporal regulation: Secondary metabolites biosynthesis may vary with temporal changes ranging from diurnal rhythms to transitional and seasonal changes (Loivamäki et al., 2007). For example, biosynthesis and emission of the monoterpenes in snapdragon (Antirrhinum majus) flowers correlate with specific expression patterns of the corresponding monoterpene synthase genes in the upper and lower lobes of flower petals during floral development, with the highest transcript levels detected at day four post-anthesis. The expression of these genes also follows a weak diurnal oscillation that is under the control of a circadian clock. (C) Spatial regulation: Secondary metabolite biosynthesis may be spatially distributed over different developmental stages or be specific to certain organs, tissues, or cell types. For example, studies on alkaloids have shown that secondary metabolic pathways can be distributed in different tissues and cell types, each one of them expressing a portion of the pathway leading to a final product. A major site for the biosynthesis of monoterpenoid alkaloids of Catharanthus roseus is the leaf epidermis, although further steps of their biosynthesis have been shown to take place in other tissues, such as palisade idioblasts, laticifers, and possibly phloem parenchyma cells (St-Pierre, Vazquez, and De Luca 1999). (D) Environmental regulation: Secondary metabolite biosynthesis is also dependent on environmental signals such as light, temperature, nutrient availability, as well as biotic and abiotic stresses (Kooke and Keurentjes 2012). For example, it is a well known phenomenon that upon herbivore or pathogen attack secondary metabolite biosynthesis increases in plant. This activation of secondary pathways is often triggered by molecules, such as jasmonate, ethylene, abscisic acid, salicylic acid, and nitric oxide, which are usually produced in defence response. Ethylene and abscisic acid are also induced by a variety of abiotic stresses, including flooding, drought, salinity, UV-B, and extreme temperatures (Zhao, Davis, and Verpoorte 2005). Metabolic engineering aims to identify suitable intervention points in these secondary metabolite pathways well in the extended network so as to enhance production of desired secondary metabolites. Up to date, genetic modifications of all major classes of secondary metabolites have
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been carried out. Interesting results have been obtained from these studies. Although expectations of dramatic product yield enhancement have not been fulfilled, these studies have developed novel insights which have added more experience to metabolic engineering field, thus, enabling researchers to better predict results in future such experiments.
1.3.2 Key components of metabolic engineering for secondary metabolites Metabolic engineering of plants for enhancing secondary metabolites requires three important components. First to inspect “Sketch” of secondary metabolite pathways i.e information generated on secondary metabolite pathway, second is selection of “Model” i.e biological experimental system and third is what genetic engineering tools should be used to bring about manipulation. 1.3.2.1
Sketch: Information available of targeted plant secondary metabolic pathway
For successful metabolic engineering of plant secondary metabolites, intervention points selected for manipulating plant secondary metabolism needs to be effective (Farré et al., 2014). This particular task is a challenging issue because complete sketch of plant secondary metabolism has not been drawn i.e complete elucidation of secondary metabolism has not been carried out yet. However, decades of research work using strategies like radio labelled tracers, precursor and inhibitor feeding etc in in vitro plant cultures has made considerable progress in elucidating secondary metabolism of plants (Sharma, Padh, and Shrivastava 2013). Further, information generated from these experiments though incomplete, encouraged researchers to carry out pioneering attempts of metabolic engineering in plant. Results generated, in turn, shed new insight in secondary metabolite pathways of plants. In simpler words, secondary metabolic pathway elucidation and engineering go hand in hand. Adding to the advantage, the past decade witnessed advent of high throughput “omic technologies” like genomics, transcriptomics, proteomics and metabolomics etc which has fuelled the process of information generation for plant secondary metabolism both at pathway level as well as network level (Vemuri and Aristidou 2005).
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A) Need of linear pathway information Biosynthetic steps catalyzed by enzymes for which corresponding structural genes are cloned obviously become intervention points of choice by metabolic engineering. In case of rate liming enzymes, rate of product formation from these biosynthetic steps can be enhanced or decreased by increasing or decreasing gene expression of cloned gene coding for rate liming enzymes using genetic engineering. It is not mere genes involved in the pathway that matter, genes of competitive pathways (which also receive carbon pool from precursor pathway along with desired pathway) may also play important role in governing carbon pool through targeted pathway. Decreasing expression of such genes could also enhance carbon pool through desired pathway. B) Need of secondary metabolite network information Multidimensional regulatory control of secondary metabolite pathways works efficiently in plants as explained in Section 3.1.2. Many research studies on phenylpropanoid pathway, terpenoid pathway and alkaloid pathways have identified numerous many regulatory steps governing them (Nagegowda 2010). With this regards, many transcriptional factors, circadian or light regulated promoter, cell or tissue specific promoters etc for many genes has been identified (Wu and Chappell 2008). Armed with this knowledge, biosynthetic steps governed by these regulatory factors can be manipulated by enhancing or decreasing expression levels of these regulatory sequences. Thus, different metabolic engineering strategies can be designed involving either structural genes coding for enzymes in pathway, or regulatory factors governing various steps of biosynthetic pathway. Unfortunately, secondary metabolite pathways as well regulatory network has not been elucidated yet. There are innumerable key players that are waiting to be discovered. Unless they are identified, effective metabolic engineering strategies cannot be designed. Therefore, the process of carrying out metabolic engineering with available knowledge needs to continue so that more knowledge is generated for future. Such knowledge will eventually aid in designing of more refined and effective metabolic engineering strategies to dramatically alter metabolite production at desired levels.
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1.3.2.2
Model: Plant based model systems
As mentioned earlier, diversity in secondary metabolites arise from three major parent scaffold: terpenoids, alkaloids and phenolics. Biosynthesis of these specialized metabolites is inherent in plants in species specific manner. Metabolic engineering usually aims to target plants which produce industrially valuable secondary metabolites. In this context, following plants have been routinely subjected to metabolic engineering attributed to the secondary metabolite pathway that they produce: Arabidopsis, maize, petunia
for
flavonoids, liginins, terpenoids, anthocyanins, lignin pathway, Papaver somniferum (opium poppy) for benzylisoquinoline alkaloid pathway (Facchini and Luca 2008) and Catharanthus roseus (Madagascar periwinkle) for monoterpene indole alkaloid pathway (Facchini and Luca 2008). A) Plant based experimental platforms Different plant based model systems have been developed using plant tissue culture technology for these plants. Metabolic engineering is carried out in these model systems under laboratory conditions. Different plant tissue culture systems include whole plant, differentiated shoot or (hairy) root cultures, dedifferentiated cell suspension or callus cultures. i) Whole Plant Agricultural science is age old well established practice. Thus, mass production of engineered plant producing desired metabolite for industry is not an issue (Wagner, Wang, and Shepherd 2004). Therefore, while considering field transfer and growth, whole plants emerge as cheaper and simpler systems to handle. However, growing transgenic plants developed from plant tissue culture technology in field requires adherence to certain strict regulatory guidelines and in some cases may be ethically unacceptable (Sharma et al., 2014). ii) Plant cell cultures An alternative to the issue is using plant cell cultures as a platform for metabolic engineering Plant cell culture allows for genetically modified plant cells to be confined in controlled manner, thus, reducing the risk of contaminating food sources and environment. Other advantages include
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growth in minimal laboratory space and production of secondary metabolites in uniform conditions. Cell cultures also provide a platform to test a metabolic engineering strategy that may later be utilized for more time-consuming production of transgenic plants (Wu and Chappell 2008). Different types of cell cultures used for metabolic engineering are as follows (a) Dedifferentiated cell cultures Metabolic engineering strategy can be tested in shortest time in callus and suspension system. However, it has its inherent disadvantages. In some cases, dedifferentiated cells have been found to lose biosynthetic capacity to produce certain metabolites. eg, vincristine is not produced in callus or suspension cells of C. roseus (Wilson and Roberts 2012). However, in case of those metabolites which are suitably produced by such cultures, it is satisfactory to apply metabolic engineering in these cultures to test strategy. Secondary metabolite production can be unstable over long periods of time in suspension cultures chiefly attributed to altered ploidy (chromosome number) levels in such cultures (Sharma, Padh and Shrivastava 2013). The tendency of plant cells to grow in aggregates can lead to development of heterogeneous subpopulations in culture where cells differ metabolically and morphologically (Kolewe, Gaurav, and Roberts 2008). Therefore, in case of metabolic engineering, it becomes difficult to screen for transformed cells, different transformed cells may give variable response to metabolic engineering diluting the effect of desired manipulation. (b) Differentiated cultures Production of some secondary metabolites requires specialized tissues or organs and therefore, capability to produce these metabolites is lost in dedifferentiated cells but not in organ cultures. In such cases, hairy root cultures are frequently used as model system for metabolic engineering. Furthermore, problem of genetic instability does not arise with hairy root cultures in comparision to dedifferentiated cultures. Chromosome number and karyotype of hairy root cultures are usually similar to those of the parent plant (Sharma, Padh, and Shrivastava 2013). However, hairy root cultures are time consuming and cannot be easily up scaled to bioreactor level compared to dedifferentiated cultures (Sharma et al., 2014). As of now, there are no reports of using shoot cultures as model systems for metabolic engineering.
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All the above factors are needed to be considered before deciding upon the plant based model system to be used depending on the pathway being analyzed. 1.3.2.3
Tool: Genetic engineering
Genetic engineering enables alteration of expression levels of genes of biosynthetic pathway in positive or negative manner. The ultimate aim is to redirect precursor pool towards desired pathway (Pickens, Tang, and Chooi 2011). Suitable intervention points can be targeted depending on the information available. These include targeted rate-liming steps, transcription regulation steps, signaling pathway steps, branching points at competitive pathway and downstream catabolic steps. Other possible intervention points can also be at special, temporal, and environmental regulatory level (Farré et al., 2014). Gene over expression, gene down regulation, genome editing etc are approaches utilized to carry out metabolic engineering in plant cell. The former two are the most commonly used techniques for the purpose. Genome editing has only recently been utilized for metabolic engineering in plants (Farré et al., 2014;Yuan and Grotewold 2015). A) Gene Overexpression Gene overexpression involves introduction of desired gene sequence under the control of strong promoter or activation signals into plant cells. The strong promoter could be constitutive or cell, tissue organ specific, or inducible under various stimuli. Increasing expression of structural gene coding for biosynthetic pathway enzymes by this means may result in increased flux through the pathway. Multigene engineering can also be carried out wherein simultaneous expression of more than one genes of pathway is carried out. Such increase of several enzyme activities can lead to enhanced flux through the pathway (Niederberger et al., 1992). For this, coding sequence of desired gene is introduced in plant cell under the control of strong promoter and/or activation sequences. Apart from structural gene, pathways specific regulators can also be over-expressed to simultaneously upregulate multiple genes involved in the pathway to enhance production of the resulting secondary metabolite (Fisher et al., 2014). By manipulating the expression of a single transcription factor, it is theoretically possible to affect the expression of several coordinately regulated biosynthetic enzymes.
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B) Gene Downregulation In addition to increasing enzyme activity or positive regulator of pathway, it may also be useful to downregulate or delete certain genes to eliminate competing pathways that may siphon off important precursors or intermediates, or simply contribute to an unnecessary use of cellular resources. RNA interference, also known as post transcriptional gene silencing (PTGS), can be used for the purpose. Double-stranded RNA (dsRNA) is the essential trigger for induction of RNA interference (RNAi). Upon perceiving ds RNA trigger, RNAi machinery activates and degrades mRNA sequences having homology to ds RNA sequence (Fire et al., 1998; Waterhouse, Graham, and Wang 1998). Biotechnology has been quick to evolve RNAi phenomenon into RNAi technology and it is now frequently used for sequence specific gene downregulation or silencing. C) Genome editing Targeted genome editing has recently emerged in plant as a potential approach to introduce specific mutations as well as to introduce one or more transgenes at particular loci. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used for the purpose. These techniques can also be used to stack multiple transgenes in plants. Another promising method based on the bacterial CRISPR/Cas system has been developed. Successful genome editing has been carried out not only in model plants but also in crops and medicinal plants (Farré et al., 2014). All these genetic engineering tools aid in applying metabolic engineering strategies in plant. However, application of these tools does not guarantee complete success of metabolic engineering strategy. It is necessary to discuss about some constraints that are unavoidable part of metabolic engineering process. They also play an important role in determining overall success of metabolic engineering process in targeted plant. Following section will dwell on the matter.
1.3.3 Constraints of metabolic engineering for plant secondary metabolites Certain constraints are associated with application of metabolic engineering strategy in plant came into light from numerous research studies on plant metabolic engineering, and therefore, are
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viewed as associated side effects of metabolic engineering process which could occur along with anticipated results. They are to be kept in mind as one of possible outcomes of metabolic engineering process when applied. These are discussed as follows 1.3.3.1
Pleiotropic effects of metabolic engineering
Manipulating secondary metabolic pathways can have often negative effects on plant growth and development. For eg. In engineering of carotenoid synthesis, the first committed enzyme, phytoene synthase (PSY), has been targeted for metabolic engineering. However, overexpression of PSY led to growth defects in tomato due to flux diversion from gibberellin synthesis (Fare et al., 1995). This example demonstrates the importance of understanding the overall impact of single-gene manipulation from systems biology perspective. This “trade off” between growth and secondary metabolite enhancement is often encountered in numerous metabolic engineering research studies ( Way et al., 2002;Wu and Chappell 2008;). Therefore, growth impairment is one of the expected possible outcomes of metabolic engineering along with desired metabolic flux enhancement. Inspite of this “trade off” metabolic engineering needs to be carried out in plants so that secondary metabolite pathways can be elucidated. 1.3.3.2
Attracting precursor pool from untargeted pathways
Efforts of increasing precursor pool through a pathway, sometimes, may bring in precursor pool meant for a secondary metabolite pathway which was not targeted. In a recent study in tobacco, overexpression of a gene upstream in plastidial volatile terpenoid biosynthesis resulted in a large increase in emitted monoterpenes and reduced levels of sesquiterpenes (Orlova et al.,2009). 1.3.3.3
Production of novel unanticipated compounds
Many research studies have reported that over-expression of a suspected-rate limited enzyme targeted can result in biosynthesis of novel end products (Wu and Chappell 2008). This observation is attributed to existence of a phenomenon called ‘silent metabolism’ in plants to explain for presence of series or group of enzymes which have no apparent endogenous substrate or function but are still found in plant (Lewinsohn and Gijzen 2009). For example, when a linalool sythase gene involved in linalool biosynthesis was overexpressed in petunia, tomato and
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carnation, in addition to the over-accumulation of linalool, these plants also produced novel metabolites linalyl-glucoside, 8-hydroxylinalool and linalool oxides respectively, depending on the silent metabolism present in that particular plant/tissue (Nagegowda 2010). Biosynthesis of novel products is an indication that partition of metabolic flux occurred amongst these compounds which was targeted only for linalool.
1.3.4 Are these constraints avoidable? Unanticipated results discussed above arise for want of thorough knowledge of secondary metabolic pathway and network. Although some preliminary components have been identified, we have just touched tip of ice berg. There may be many more players involved in regulation machinery of secondary metabolite biosynthesis. There are innumerable components waiting to be discovered. Until that happens, accurately predicting the cellular response of any metabolic perturbation is an extremely complex and difficult procedure. Therefore, if metabolic engineering for secondary metabolites in plants has to be successful, continued efforts are needed in direction of secondary metabolic pathway elucidation and identification of suitable intervention points. In this view, the present study was designed aiming to analyze regulatory role of crosstalk between MIA and phenylpropanoid pathway on precursor flow in MIA pathway in model plant C. roseus.
1.4. Towards Metabolic Engineering in C. roseus: The Project 1.4.1 Catharanthus roseus Catharanthus roseus (G. Don), (Apocynaceae family), is a well known medicinal plant, being source of two important pharmaceutically active anticancer compounds namely vinblastine, vincristine (van Der Heijden et al., 2004) (Figure 1.4). These compounds have multimillion dollar market. The plant also produces many other pharmacologically active MIA compounds of pharmaceutical interest eg ajmalicine and serpentine [both antihypertensive with high commercial value (van Der Heijden et al., 2004). All these compounds are part of the secondary
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metabolism of the plant, and arise from monoterpenoid indole alkaloid (MIA) biosynthetic pathway (Miettinen et al., 2014). The importance of these phytochemicals has motivated researchers to carry out extensive studies to elucidate MIA biosynthetic pathway and regulation since four decades now. The idea is to use this information to implement a suitable metabolic engineering approach like genetic manipulation by overexpression and/ or downregulation of suitable gene/s for metabolic engineering as it is considered most promising strategy to effectively manipulate MIA pathway on permanent basis as desired (Zhao and Verpoorte 2007).
Figure 1.4: Catharanthus roseus plant
1.4.2 C. roseus secondary metabolism: General overview As previously discussed, diverse secondary metabolites arise from a three major chemical scaffolds: phenolic, alkaloids and terpenoids. For all higher plants including C. roseus, the shikimic acid pathway is an entry point into to aromatic secondary metabolism. Chorismic acid is the end product of the pathway giving rise to aromatic amino acid phenyl alanine and tryptophan which in turn give rise to phenylpropanoids and indole alkaloid pathway, respectively. Phenylpropanoid pathway further has following branches i.e flavonoid, anthocyanin and lignin
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biosynthetic branches. Terpenoid pathway arises from mevalonic acid (MVA) pathway and or 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Pathway branching described uptil now is conserved in all higher plants. Specialized pathway branching with respect to C. roseus begins when terpenoid pathway further branches into secoirridoid pathway. Then indole alkaloid pathway and secologanin pathway combine to form monoterpene indole alkaloid (MIA) pathway which is characteristic highlight of C. roseus secondary metabolism. An overview of general scheme of secondary metabolite pathways in C. roseus is shown in Figure 1.5. 1.4.2.1
MIA pathway
MIA pathway in C. roseus is a complex pathway including at least 30 coordinately regulated enzymatic steps producing at least 35 known intermediates. The pathway is spread over at least 4 different cell types and in these cells at least 5 different subcellular compartments are involved (O’Connor and Maresh 2006). Up to now, 30 biosynthetic and 4 types of regulatory genes have been cloned and identified (Pan et al., 2012). However, major portion of MIA biosynthesis remains to be unveiled. The terpenoid part starts from geraniol produced by the MEP pathway. The indole part derives, via anthranilate, from chorismate which is a major branching point in the shikimate pathway. Terpenoid pathway gives rise to secologanin and indole pathway gives rise to trypramine. Both these metabolites combine to give rise to strictosidine which is general precursor for MIAs. Strictosidine then gives rise to other downsttream MIAs like ajmalicine, cathranthine, vindoline. Cathanranthine and vindoline then combine to give rise to bisindole alkaloids vinblastine followed by vincristine. Initially MIA metabolic pathway elucidation studies were initiated in dedifferentiated (callus and suspension) cultures as model systems. Then it was realized that dedifferentiated cultures do produce early MIAs like catharanthine, ajmalicine, serpentine etc but lack biosynthetic capacity study to produce late MIAs like vindoline, vinblastin and vincristine (St-Pierre, Vazquez, and De Luca 1999). Therefore, these cultures have been good model systems to elucidate early MIA pathway genes and regulation. Differentiated cultures can mimic the native tissue complexity of the plant. Therefore, differentiated hairy root cultures of C. roseus are known to produce late stage MIA genes. These cultures can be used as model systems
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to study late stages or entire of MIA pathway (Moreno-Valenzuela et al., 1998) as depicted in Figure 1.4.
Figure 1.5: Overview of general scheme of secondary metabolism of in C. roseus. Broken arrows indicate multiple enzymatic reactions.Important pathways and their cloned key genes are shown. PAL Phenylalanine ammonia lyase, CM Chorismate mutase, G10H Geranyl 10 hydroxylase AS Anthranilate synthase, TDC Tryptophan carboxylase, SLS secologanin synthase, STR Strictosidine synthase, SG Strictosidine glucosidase
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1.5 Genesis of Project Phenyl propanoid and MIA pathway crosstalk in C. roseus As discussed earlier, Phenylpropanoid pathway utilizes chorismate which is also precursor for MIA pathway and, thus could be partitioning off significant carbon pool from the MIA pathway at its origin itself (Mustafa and Verpoorte 2007). Evidence for cross talk Phenylalanine ammonia lyase PAL enzyme is the first and key rate-limiting enzyme of phenylpropanoid pathway (Ferrer et al., 2008). PAL enzyme produces trans cinnamic acid from amino acid phenylalanine. Cinnamic acid feedback inhibition of PAL enzyme in C. roseus cell suspension led to marked increase in biosynthesis of MIA (Godoy-Hernández and Loyola-Vargas 1991). Other phenolics like caffeic acid and ferulic acid have been found to enhance the growth and total alkaloid content in C. roseus plants (Mustafa and Verpoorte 2007). Recently, overexpression of a regulatory gene (ORCA3) and a structural gene (G10H) in the MIA pathway resulted in increases of MIA (strictosidine, vindoline, catharanthine and ajmalicine) accumulation in C. roseus plants along with feedback on phenylpropanoid biosynthesis (Pan et al., 2012).Thus, a possible competition for carbon pool exists between both the pathways. The potential influence of this crosstalk on regulating metabolic flux through the MIA biosynthetic pathway has remained underexplored and needs to be analyzed. All these evidences suggest phenylpropanoid pathway downregulation could have significant regulatory role on metabolic flux tight regulation in MIA pathway. PAL enzyme is the first and key ratelimiting enzyme for phenylproapnoid pathway. Downregulation of phenylpropanoid pathway has the potential to enhance biosynthesis of MIAs in C. roseus resulting in enhanced yield. Detailed analysis of this crosstalk would benefit the present knowledge available for MIA pathway. With this propspectus in mind, the present project proposes metabolic engineering in C. roseus using RNAi approach to downregulating expression of PAL enzyme.
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1.5.1 Strategy Shikimic acid pathway produces chorismic acid which in turn produces amino acid precursors phenylalanine and tryptophan for both phenylpropanoid pathway and MIA pathway, respectively. PAL enzyme is the first and key rate-limiting enzyme of phenylpropanoid pathway which catalyzes conversion of cinnamic acid from amino acid phenylalanine (Ferrer et al., 2008). Cinnamic acid precursor then gives rise to all other diverse phenylpropanoids. The strategy of the present project is to decrease precursor pool (i.e cinnamic acid substrate) flow towards phenylpropanoid pathway by downregulating PAL enzyme expression using RNAi phenomenon in dedifferentiated cultures of C. roseus. Downregulation of PAL gene expression has potential to redirect carbon flux towards MIA pathway. Chorishmic acid
Common precursor pool
RNAi of PAL
X
? Increase in carbon flux
Phenylpropanoid pathway
MIA pathway
1.6 Objectives To prove our hypothesis of cross talk following objectives were set for study 1) Establishment and maintenance of in vitro culture of C. roseus 2) Design and development of suitable PAL RNAi sequence 3) Design, development and construction of RNAi expressing vector. 4) Genetic transformation of Agrobacterium with RNAi expression vector 5) Agrobacterium mediated genetic transformation of C. roseus cells. 6) Analysis of transformants
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