Neem, or Azadirachta indica, belongs to the family Meliaceae. It is native to India and Burma and has since been introduced to several nations in Africa and North America. A. indica holds significant importance in traditional medicine systems, particularly in India and other parts of South Asia (Kumar and Navaratnam, 2013; Moga et al., 2018; Blum et al., 2019).

Various biological actions have been discovered in A. indica, and it has been investigated for its possible antibacterial, antiviral, antifungal, molluscicidal, and antihyperglycemic characteristics (Ufele et al., 2013; Ezeigwe et al., 2015; S. Abdelhady et al., 2015; Ashfaq et al., 2016; Joy Sinha et al., 2017; Osman Mohamed Ali et al., 2017; Altayb et al., 2022).

Furthermore, the SARS-CoV-2 (COVID-19) pandemic (Kalasariya et al., 2022) has lately posed a challenge to humanity, and different compounds have been investigated in silico to treat the disease. Docking investigations of A. indica molecules have also provided encouraging results for their inhibitory action against various illnesses such as SARS-COV-2, malaria, and dengue (Lavanya et al., 2015; Dwivedi et al., 2016; Khanal et al., 2019; Adegbola et al., 2021; Baildya et al., 2021).

The A. indica has been thoroughly studied for its secondary chemical compounds and for its potential application in the discovery and synthesis of triterpenes, which are among the most abundant and highly complex families of plant-derived natural products.

A predominant focus of research in A. indica is the presence of an important secondary metabolite compound known as azadirachtin, which is a triterpenoid class of limonoids. Azadirachtin, the principal insecticidal component contained in the kernel of Neem seeds, displays high bioactivity against different kinds of insects (Schmutterer, 1995; Schmutterer and Singh, 1995; Hummel et al., 2015).

Growing concerns about the potential negative impacts of chemical pesticides on human health, the environment, and non-target organisms have led to an increasing preference for alternative crop protection methods (Ajiboye et al., 2022). Consequently, there is a greater focus on the development and utilization of plant- or microbe-based biopesticides that are both bioactive and biodegradable. Azadirachtin-based pesticides are environmentally friendly, biodegradable, and non-toxic to wildlife, plants, and birds (Raizada et al., 2001; Kilani-Morakchi et al., 2021). Azadirachtin has shown very minimal toxicity to mammals and has great selectivity for its target organisms (Mordue (Luntz) et al., 2005; Amaral et al., 2019). Azadirachtin is the predominant compound responsible for controlling of insects in agriculture (Vacante and Bonsignore, 2018). Over the past three decades, there has been an increase in the utilization of Neem-based insecticides, primarily attributed to the discovery and isolation of azadirachtin, the key bioactive compound responsible for its insecticidal properties (Chaudhary et al., 2017; Pasquoto-Stigliani et al., 2017). Azadirachtin has been successfully commercialized, and it is still widely accepted as being the most effective botanical pesticide that is in use in agricultural production all around the world (Isman and Grieneisen, 2014; Chaudhary et al., 2017; Aribi et al., 2020).

The biosynthesis route of Neem, which is known to synthesize physiologically and economically relevant triterpenoids with extraordinarily complex carbon skeletons and diverse functional groups, is of great interest among researchers. The first successful synthesis of azadirachtin took 20 years to complete (Jauch, 2008; Veitch et al., 2008) and comprises 71 steps; however, the yield is merely 0.00015%, and thus the production of azadirachtin at the industrial scale is not feasible.

The recent identification and functional characterization of genes involved in the formation of these triterpenoid precursors, which are responsible for the synthesize of limonoids, was made possible by studies in transcriptomics and genomics. Thus, omics research offers a useful technique for examining the biosynthesis of secondary metabolites. While the biochemical constituents of Neem have been widely investigated, its genetic, molecular, and genomic resources are scarce.

The ability of Meliaceae plants to metabolize structurally diverse and physiologically relevant compounds is well established (Lin et al., 2022). The massive amount of literature available across several platforms makes it challenging to find information on each Neem metabolite. Azadirachtin, the most researched, has a challenging chemical structure that belongs to the tetranortriterpenoid class and is present in several forms, the most well-known of which are azadirachtin A, and azadirachtin B ((EFSA) et al., 2018; Fernandes et al., 2019).

The secondary metabolites found in various parts of the tree endow Neem with an array of biological capabilities. Azadirachtin, Azadirone, Gedunin, Nimbin, Salannin, and Vilasinin are some of the major metabolites known to exhibit substantial pesticidal and/or therapeutic properties (Dhar et al., 1998; Isman, 2006; Pravin Kumar et al., 2007; Boursier et al., 2011; Gupta and Diwan, 2017). Out of the several limonoids, azadirachtin accounts for most of its metabolite pool. Table 1 shows the structures and properties of a few secondary metabolites.

According to Verpoorte and Alfermann (2000), there are three main classes of secondary metabolites that may be distinguished from one another based on their biosynthetic pathways. These classes include terpenoids, polyketides, and phenylpropanoids (Verpoorte and Alfermann, 2000). Two major metabolic pathways—mevalonate (MVA) and methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP)—are used by plants for synthesizing terpenoids (Shi et al., 2010).

In higher plants, the traditional mevalonate pathway mostly produces the precursors, which are essential for the synthesis of sesquiterpenes, triterpenes, and sterols in the cytosol and mitochondria, while the hemi-, mono-, sesqui-, and diterpenes are produced by the non-mevalonic acid pathway.

Although the azadirachtin biosynthesis in Neem is not well established, the initial step in triterpenoid biosynthesis involves the cyclization of 2,3-oxidosqualene, which is catalyzed by oxidosqualene cyclase ( Figure 1 ). This cyclization reaction represents the primary diversification level in the biosynthesis of triterpenoids (Abe et al., 1993). In addition to this, tirucallol (C30 Triterpene), a steroid of triterpenoids, is a potential precursor of Neem azadirachtin biosynthesis (Ley et al., 1993; Johnson et al., 1996; Ley et al., 2008).

In A. indica, two different levels of biochemical complexity are assumed to be involved in the production of azadirachtin from tirucallol (Hansen et al., 1994; Ley et al., 2008). Initially, a reduction of four atoms occurs in the lateral chain (Ley et al., 1993; Dewick, 2002), followed by the cyclization of the residual atoms to generate a furan ring. This leads to the formation of limonoids, namely azadirone and azadiradione. Following this, the C-ring undergoes an opening process, leading to the generation of C-seco-limonoids, namely nimbin, and salannin, and the third ring of apotirucallol is oxidized (Ley et al., 1993; Johnson et al., 1996; Puri, 1999; Ley et al., 2008). Additional rearrangements and oxidations are necessary to produce azadirachtin, which is classified as one of the most extensively oxidized triterpenoids (Aerts and Mordue, 1997).

Omics is a potent tool for identifying essential genes for significant traits, clarifying physiological event mechanisms, and revealing unknown metabolic pathways. A whole genome sequence provides a complete overview of how the functional elements of the genome are structurally organized. These structural elements carry the knowledge of the evolutionary history of an organism (Subramanian et al., 2020).

In addition to genomes, transcriptomes have also been shown to be essential in deciphering the molecular mechanisms and metabolic pathways underpinning a wide range of biological functions. High-throughput sequencing technologies such as RNA sequencing (RNA-seq) are utilized to create extensive transcriptome atlases, and these technologies also contribute to a better knowledge of the functional components that make up the genome of any species (Jiang et al., 2015).

Considering the advantages of omics technology can aid in identifying the unexplored pathways across different species, and the integration of transcriptome data with other omics approaches, such as proteomics and metabolomics, can provide a more comprehensive understanding of the biology of any given species (Yan et al., 2022). By correlating gene expression with protein abundance and metabolite levels, researchers can unravel the complex interactions and regulatory networks underlying physiology and biochemistry.

In the case of A. indica, the biosynthetic pathway for triterpenoid production was not well studied until the publication of the first genome and transcriptome of A. indica. These studies helped in identifying the repeat elements, the nucleotide composition of nucleotides, and expression profiles of initial genes involved in terpenoid production in different tissues of Neem. A. indica was the first Meliaceae family member to be sequenced genome-wide (Krishnan et al., 2012).

The Relative expression of HMGR (HMG-CoA reductase) was higher when compared to the MEP pathway, confirming that the Mevalonate pathway might contributes to the isoprene units of triterpenoids. The distribution of limonoids varies across different tissues reported (Pandreka et al., 2015; Aarthy et al., 2018).

Some of the critical enzyme like oxidosqualene cyclase (AiOSC1) involved in the pathway for the synthesis of triterpenoid was reported by (Hodgson et al., 2019), but they failed to mention triterpene synthase (TTSs), including tirucalla-7, 24-dien-3β-ol synthase, which were functionally characterized by different researchers (Pandreka, 2018; Thulasiram et al., 2018). These Triterpene Synthases (TTSs) work on 2,3-oxidosqualene to create cyclic compounds, which is the crucial step for steroid and triterpenoid biosynthesis (Volkman, 2005). Later, Triterpene synthase (TTS1), was also functionally characterized by a team led by Pandreka (Pandreka et al., 2021).

These clusters of genes mentioned by the various authors from 2012 to 2022 have paved the way for the identification of genes responsible for the production of industrially and medically important triterpenoids in A. indica. The integration of omics, bioinformatics, and genetic engineering technologies holds a great deal of promise for expanding our understanding of the process and locating any missing links in the chain of events that led to the creation of azadirachtin (Kumar et al., 2021). Although there aren’t any well-established genes involved in the numerous processes leading from tirucallol to azadirachtin, utilizing these methodologies can nevertheless yield really helpful insights.

Researchers are able to conduct an exhaustive investigation of the genetic and molecular components that are associated with a pathway by making use of omics tools such as genomics, transcriptomics, and proteomics. Tools and techniques from the field of bioinformatics can be used to assist in the processing and interpretation of huge amounts of biological data, which can in turn facilitate the discovery of candidate genes and probable enzymes involved in this class of triterpenoids.

The discovery of the missing link in the biosynthesis of azadirachtin in A. indica presents a significant difficulty because the biosynthesis of azadirachtin involves a complicated network of processes, with metabolites serving as both substrates and products and gene products acting as enzymes that catalyze the appropriate reactions. Once the underlying molecular mechanisms are understood, new opportunities arise for altering and optimizing the production of azadirachtin, which may result in increased yields or novel applications in the pharma and agriculture industries.

A. indica is classified as a high-value medicinal tree and a great source for azadirachtin, which is known for its insecticidal activity. Along with these molecules, A. indica also synthesizes several other biologically active compounds with demonstrated pharmacological attributes. Despite extensive research on A. indica’s enzymatic pathway machinery, the downstream genes responsible for azadirachtin production have not yet been identified. This knowledge gap highlights the need for in-depth studies that employ an integrated omics approach. By combining various omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, researchers can gain a comprehensive understanding of the biosynthesis of different alkaloids and their analogs present in A. indica. Identifying and isolating these compounds is crucial for their industrial and pharmacological applications. With the help of omics tools, the biosynthesis pathways of several bioactive substances in A. indica can be mapped out and can provide more useful genetic information about A. indica. The potential of omics research in A. indica appears to be promising, presenting prospects for enhancing our understanding of Neem’s biosynthesis pathways, identifying new bioactive compounds, evaluating genetic variability, expediting functional gene discovery, directing breeding endeavors, and implementing systems biology methodologies. These advancements can have significant implications for the development of Neem-based applications in the medical, agricultural, and industrial sectors.

ND: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing- original draft. AI: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing- original draft. MP: Data curation, Formal analysis, Investigation, Methodology, Software, Writing- review & editing. TK: Conceptualization, Funding acquisition, Project administration, Resources, Software, Supervision, Writing- original draft. VY: Conceptualization, Project administration, Supervision, Visualization, Writing- original draft, Writing- review & editing. DS: Conceptualization, Funding acquisition, Project administration, Resources, Software, Supervision, Writing- original draft. AP: Conceptualization, Project administration, Supervision, Visualization, Writing- original draft, Writing- review & editing.