Biosynthesis and classification

Flavonoid biosynthesis occurs through the phenylpropanoid pathway; this is a major metabolic route to produce secondary metabolites that support plant growth, structural integrity and responses to environmental stress [14]. This pathway occurs mainly in the cytosol and involves the coordinated action of multi-enzyme complexes; this tight regulation prevents the accumulation of reactive and potentially toxic intermediate metabolites in the cytoplasm [15].

The biosynthetic pathway (Figure 1) begins with the conversion of phenylalanine to p-coumaroyl-CoA through the sequential action of three key enzymes, such as phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL) [16].

Figure 1.: Flavonoid biosynthesis pathway

This initial step removes the amino group from phenylalanine to produce the phenylpyruvate intermediate. The first enzyme activated in flavonoid biosynthesis is chalcone synthase (CHS). It catalyses the formation of chalcone by condensing one molecule of p-coumaroyl-CoA with three molecules of malonyl-CoA [17,18]. As the key precursor, chalcone is then converted to flavanone by chalcone isomerase (CHI) via an intramolecular cyclization reaction [19]. CHI enzymes are classified into four types. Type I CHI is present in most land plants and catalyses the conversion of naringenin chalcone into (2S)-naringenin. Type II CHI is specific to leguminous plants and catalyses the formation of 5-deoxyflavanones such as liquiritigenin [20]. Type III proteins resemble CHI structurally but function as fatty acid-binding proteins, while type IV CHI-like proteins (CHIL) assist CHS in chalcone formation [21,22].

Following flavanone formation, flavanone 3-hydroxylase (F3H) converts flavanones to dihydroflavonols as key intermediates for the biosynthesis of flavonols, anthocyanins and proanthocyanidins [23]. Additional hydroxylation reactions on the B-ring are catalysed by cytochrome P450 enzymes such as flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), leading to structural diversity among flavonoids [24]. Different branches of the pathway lead to distinct flavonoid subclasses. Flavone synthase (FNS) transforms flavanones to flavones, while isoflavone synthase (IFS) redirects flavanones into the isoflavone pathway, which is particularly important in leguminous plants [25]. Isoflavone biosynthesis involves several additional enzymes, resulting in a wide range of isoflavone derivatives with diverse biological functions. In some plants, alternative cyclization reactions catalysed by stilbene synthase (STS) produce stilbenes instead of flavonoids. This branch pathway occurs only in specific species such as grapevine, pine, sorghum and legumes. Other specialized enzymes, such as aurone synthase (AURS), generate aurones, yellow pigments commonly found in flowers and involved in pollination [26,27].

After synthesis in the cytosol, flavonoids are transported into the vacuole, where they are stored and stabilized. This transport process requires membrane permeability and tight regulation to ensure vacuolar sequestration and metabolite stability [28]. There are three distinct flavonoid transport mechanisms: vesicle trafficking, membrane transporters and glutathione S-transferase (GST)-mediated transport [29]. In brief, flavonoid transport pathways can be classified as follows [30-32];

• Proton-dependent transporters, in this mechanism, a proton gradient between the cytosol and the vacuole (or cell wall), generated by H+-ATPases and H+-PPases, is thought to drive flavonoid transport. Once inside the vacuole, the acidic pH and flavonoid acylation promote conformational modification, allowing the formation of stable and functional metabolites.
• ATP-binding cassette (ABC) transporters and multidrug and toxic compound extrusion (MATE) transporters: ABC transporters mediate direct, energy-dependent transport of flavonoids into the vacuole; thus, they prevent the accumulation of potentially toxic intermediate metabolites [26]. These proteins couple ATP hydrolysis with substrate translocation across membranes, often via conjugation with glutathione (GSH) catalysed by glutathione S-transferase (GST). MATE transporters exhibit substrate specificity due to structural mutations. Although their exact mechanism is not fully understood, MATE transporters are believed to play roles in detoxification and disease resistance signalling.
• Glutathione S-transferase (GST)-mediated transport: In this pathway, flavonoids bind to GST as a carrier protein to facilitate their transport to the vacuole. GSTs are also associated with cellular membranes of the endoplasmic reticulum (ER) and vacuole, particularly in plant cells that produce high levels of anthocyanins.

Flavonoids are characterized by a basic 15-carbon flavone backbone (C6-C3-C6) with two benzene rings (A and B) connected by a three-carbon pyran ring. Based on the variations in the C3 unit, flavonoids are further divided into subclasses, including flavanones, isoflavones, anthocyanins, chalcones, dihydrochalcones, flavanols and proanthocyanidins [33]. Furthermore, according to structural modifications of the core skeleton, flavonoids are commonly classified into seven major subclasses (Table 1) [34,35]. In nature, flavonoids occur in two main forms: glycosides and aglycones [36]. Glycosylation in natural products involves the attachment of sugar moieties through carbon-carbon or carbon-oxygen bonds, resulting in more stable metabolites through the interaction between secondary metabolite acceptors and sugar groups [37].

Subclass | Key structural feature | Ring B attachment | C2-C3 bond | Common aglycones
Flavones | C4 ketone group; lack of C3 hydroxyl | C2 | Double | Apigenin, luteolin
Flavonols | 3-hydroxyflavones; hydroxyl group at C3 | C2 | Double | Quercetin, kaempferol
Flavanones | Dihydroflavones; lack of C2-C3 double bond | C2 | Single | Naringenin, hesperetin
Isoflavonoids | B-ring migration from C2 to C3 | C3 | Double | Genistein, daidzein
Flavanols | Flavan-3-ols; C3 hydroxyl, no C4 ketone | C2 | Single | Catechin, epicatechin
Anthocyanins | Flavylium ion (chromenylium) skeleton | C2 | Double | Cyanidin, pelargonidin
Chalcones | Open-chain; α, β unsaturated carbonyl | N/A | N/A | Isoliquiritigenin, phloretin

Among secondary metabolites, flavonoid production in plants is highly limited and varies by plant species, plant parts, age and environmental conditions. The very low levels of plant flavonoid production pose a challenge for research on these secondary metabolites. Studies have shown that flavonoid production can be enhanced through several approaches, including triggering production-inducing factors, regulating enzymes involved in flavonoid biosynthesis and utilizing microorganisms[38-40]. Plant metabolic engineering is carried out through the overexpression of key enzymes that actively participate in flavonoid biosynthetic pathways [41]. In addition, regulation of transcriptional and translational mechanisms using transcription factors, target gene expression [42] and prevention of end-product toxicity in plants [43] can also be implemented. Furthermore, the use of microorganisms for flavonoid production has gained considerable attention, particularly because of its low energy requirements, high product purity and low emissions of waste such as sulfates, nitrates, or nitrites [44]. For example, microorganisms such as Escherichia coli and Saccharomyces cerevisiae can produce naringenin and apigenin [45-47]. The de novo production of flavonoids by microorganisms can yield certain flavonoids at gram-per-liter fermentation levels, which are significantly higher than those in natural plant production. For comparison, the naringenin content in grapefruit (Citrus paradisi) was 16.90 mg per 100 mg [48], whereas the use of Corynebacterium glutamicum was able to produce 35 mg L-1 naringenin and 37 mg L-1 eriodictyol [49] and Escherichia coli produced 155 mg L-1 naringenin [50].