Limonoid biosynthesis in azadirachta indica: characterization of pathway genes and analysis of labeled metabolites through stable isotope feeding

Abstract

Due to their immobile nature, plants have evolved and adapted to different environmental stresses by effective synthesis, storage and emission of secondary metabolites in the necessary time and space. These secondary metabolites or natural products are of immense fortitude to mankind due to its different kind of ameliorating property on human health ailments. Neem tree, one of the Indian medicinal plant well-known and being used for its healing property from ancient past is of profound interest and hence, the study of its biosynthetic pathway, understanding its localization, regulation etc., will pave the way to design an efficient way of treating human diseases as a natural means of medicament. The thesis entitled, “Limonoid biosynthesis in Azadirachta indica: Characterization of pathway genes and analysis of labeled metabolites through stable isotope feeding” is concerned with the study of biosynthesis of limonoids and other secondary metabolites and their localization in neem.

Neem tree is known for its bountiful and diverse natural products conferring numerous medicinal and agricultural uses for mankind1. Among these plenty of natural products, most of them belong to triterpenoid (limonoids)2. More than 150 limonoids have been isolated and characterized from neem so far and each of them has been studied for its biological activities3. Azadirachtin, one of the highly complex limonoid had been studied widely for its excellent anti-feedant, insecticidal property, with no proven harmful effects on vertebrates and are found to be an unsurpassable natural product identified hitherto for its efficiency. The successful chemical synthesis of this complex molecule was accomplished after 22 years of efforts by Ley et al.4, 5. Azadirachtin and other bioactive limonoids are highest in the fruits of neem tree. There is an indispensable requisite for metabolic engineering of this pathway in heterologous system. Therefore, the study of azadirachtin biosynthetic pathway is highly essential to harness the biosynthetic capacity of the tree for its medicinal properties. Following are the main goals of the thesis, To identify and characterize the organelles/ intra-cellular compartments involved in the storage of limonoids in different tissues. • To trace the flow of carbon in limonoid skeleton through stable isotope feeding experiments. • To study the expression level of the genes involved in isoprene biosynthetic pathway and to characterize the putative candidate genes associated with limonoid biosynthesis.

Development of in vitro cultures of neem, limonoid profiling and study of organelles and cells involved in limonoid storage In our laboratory, we have identified previously, the tissue and developmental stage specific differential distribution and abundance of ring intact (basic) and C-seco limonoids in different neem tissues6. This incredible and impressive abundance of limonoids in fruit and leaf tissues actuated us to look for the specialized cells involved in accumulating limonoids and also the development of neem plantlets, in vitro callus from kernel and callus derived suspension system to study limonoid biosynthesis. C-seco limonoid group exists as major limonoid synthesized in the in vitro plantlets and also callus and suspension cells. In the present study, histochemical analysis of different tissues has been carried out to study the limonoid localization in cellular and subcellular compartments. It suggested that laticifers are rich in basic limonoids and oil bodies are found to be the major source of C-seco-limonoids in pericarp and kernel of neem fruit respectively, whereas limonoids exist in abundance in idioblast vacuoles in pericarp and leaf tissues. Average number of idioblasts per mm2 of tissue was comparatively high in pericarp to that of leaf, which matched with the abundance of limonoids in these tissues respectively

Ex vivo tracer study of limonoid biosynthesis in Azadirachta indica Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. A well-known potent anti-feedant, highly complex molecule, azadirachtin and many other biologically active limonoids are distributed in different tissues of neem7. The biosynthetic machinery that plant cell employs for the production remains unexplored for this wonder tree. Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using 13C labeled isotopologues of glucose in neem cell suspension. In order to identify the signature fragment of azadirachtin A generated through ESI-MS, fragments generated from MS/MS of azadirachtin derivatives such as azadirachtin B, H, 3-deacetylazadirachtin A, epi-azadirachtin D, vepaol were studied comparatively at different normalized collision energies. The plausible fragmentation pathway for azadirachtin A has been proposed showing the structure-fragment relationship. This helped in tandem mass spectrometry (MS/MS) analyses of labeled limonoid.extract,which lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis (Figure 2). Therefore, the isoprene units contributed for limonoid skeleton biosynthesis are found to be formed through mevalonate (MVA) pathway (Figure 3).

Study of the role of MVA and MEP pathway in limonoid biosynthesis and characterization of pathway genes To study the role of MVA and MEP pathway towards limonoid biosynthesis, chemical inhibition of the pathways along with concomitant 13C Glc labeling was carried out. Treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition (Figure 4). This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells as evident from real-time PCR analysis (Figure 5). This chapter also deals with the cloning of putative acyltransferase genes in neem. Neem limonoids being triterpenoids are biosynthesized from the triterpene intermediate squalene, which en route to 2,3 oxidosqualene leading to cyclized triterpene intermediate which further modifies to form protolimonoids. Limonoids further undergoes numerous functionalizations such as redox reactions, hydroxylation, acetylation, tigloylation etc. to form several limonoids in multistep pathway, which are yet to be elucidated. In order to understand limonoid diversity in neem, putative acyltransferase ORFs were selected from neem transcriptome for cloning. The selected acyltransferases matched with BAHD family of plant acetyltransferases, which transfers group from CoA to natural products. The two full-length ORFs were cloned into bacterial and yeast vector systems and characterization of the expressed protein to understand its function with limonoids and/ other terpene or non-terpene alcohols as substrates.