2. Biosynthetic Pathway of SMs

The precursors of metabolites are essentially produced in the Krebs cycle and shikimate pathway. The proposed SM biosynthesis pathway is shown in Figure 2. Primary metabolites are the critical precursors of SMs. Primary and SMs can be distinguished based on their chemical structure, function, and distribution in plants. The fundamental biosynthetic pathways of metabolites are conserved in the majority of plants, with most primary metabolites found in every tissue type. The maintenance of this metabolic core has led to the occurrence of a limited number of fundamental metabolic frameworks. Frequent glycosylation, methylation, hydroxylation, acylation, oxidation, phosphorylation, and prenylation, as well as fewer chemical alterations due to tailoring of enzymes, causes a wide range of modifications in basic structures. Based on biosynthesis pathways, SMs can be divided into three main groups: phenolic compounds synthesized in the shikimate pathway, terpenes synthesized in the mevalonic pathway, and nitrogen-containing compounds synthesized in the tricarboxylic acid cycle pathway ^[7].

Figure 2. Schematic of the secondary metabolite biosynthesis pathway.

Shikimic acid, the precursor of the shikimate pathway, is produced from a combination of phosphoenolpyruvate (from the glycolytic pathway) and erythrose 4-phosphate (from the pentose phosphate pathway). Phenylalanine, tyrosine, and tryptophan are produced in the shikimate pathway; these are the building blocks of protein synthesis and common precursors for plant SMs such as phenolics and nitrogen-containing compounds ^[6]. Phenylalanine is the common precursor of flavonoids, lignans, lignins, condensed tannins, and phenylpropanoid/benzenoid volatiles; tyrosine further produces isoquinoline alkaloids, pigment betalains, and quinones (e.g., tocochromanols and plastoquinone); and tryptophan is the precursor of alkaloids, phytoalexins, indole glucosinolates, and the plant hormone auxin ^[8].

Seven steps are involved in the shikimate pathway to generate the end-product chorismate. In the first step, phosphoenolpyruvate and erythrose-4-phosphate are condensed to 3-deoxy-o-arabino-heptulosonate 7-phosphate (DAHP), which can formally be considered a 2-deoxy-D-glucose-6-phosphate derivative. In the second step, DAHP is exchanged with 3-dehydroquinate synthase to form the highly substituted cyclohexane derivative 3-dehydroquinate. The remaining steps in the shikimate pathway serve to add side chains and two of the three double bonds that convert this cyclohexane into a benzene ring (the hallmark of aromatic compounds). The third and fourth reaction in the shikimate pathway includes the dehydration of dehydroquinate to 3-dehydroshikimate, which is catalyzed by 3-dehydroquinate dehydratase (DHD), and the reversible reduction of 3-dehydroshikimate into shikimate using NADPH, which is catalyzed by shikimate dehydrogenase (SDH). DHD and SDH not only catalyze these respective reactions but are also part of the AROM complex in fungi. Furthermore, they are monofunctional in Escherichia coli but bifunctional in plants in the fused form of the DHD–SDH enzyme. The fifth step of the shikimate pathway is the formation of shikimate 3-phosphate. In this step, shikimate kinase catalyzes the phosphorylation of shikimate at the C3 hydroxyl group using ATP as a substrate. The sixth and seventh steps are catalyzed by 5-endolpyruvylshikimate 3-phosphate synthase (EPSP) and chorismate synthase, respectively. EPSP (also known as 3-phodphoshikimate 1-carboxyvinyltransferase) regulates the next to last step of the shikimate pathway by transferring the enopyruvyl moiety of phosphoenolpyruvate into shikimate 3-phosphate. Chorismate synthase is the final enzyme to participate in the shikimate pathway; it catalyzes the conversion of EPSP into chorismate, which is the precursor of SMs. In this final step, 1,4-anti-elimination of the 3-phosphate and C6-pro-R hydrogen from EPSP introduces the second double bond in the ring to produce chorismate. In higher plants, chorismate is the precursor of tryptophan, tyrosine, phenylalanine, salicylate, phylloquinone, and folate; it is regulated by enzymes such as chorismate mutase, iso-chorismate synthase, anthranilate synthase, and aminodeoxychorismate synthase ^[8].