**3.1 Phenylpropanoid pathway**

Several flavonoids are synthesized in plants using the phenylpropanoid pathway from naringenin chalcone. A recently established biosynthesis pathway was established in a heterologous microorganism by fermentation of *E. coli* carrying an artificially assembled phenylpropanoid pathway to produce flavanones from amino acids such as phenylalanine and tyrosine [42]. Plants use phenylalanine ammonia lyase (PAL) to deaminate phenylalanine to produce cinnamic acid as the first step in the phenylpropanoid pathway. As a result of the action of cinnamate-4-hydroxylase (C4H), cinnamate-4-hydroxylase (C4H) converts cinnamate to p-coumaric acid, which is then converted to *p*-coumaroyl-CoA by 4-coumarate: CoA ligase, cinnamic acid becomes p-coumaric acid. The naringenin chalcone is synthesized by three acetate units from malonyl-CoA with p-coumaroyl-CoA using Chalcone Synthesis (CHS). In vitro, naringenin is converted to naringenin using chalcone isomerase (CHI) or nonenzymatically without activating enzymes [43].

#### **3.2 Enhancement of flavonoid synthesis**

For heterologous flavonoids production, many molecular biology technologies are used, including choosing promoter and target genes, knocking out related genes, over expressing malonyl-CoA, and creating artificial P450 enzymes. Genes from the phenylpropanoid pathway are cloned in the host under the control of the promoter, due to which secondary metabolites are often expressed heterologously. In an effort to promote flavonoids production, several promoters have been used depending on host requirements, including T7, ermE, and GAL1 promoters [41]. One of the limitations of microbiological flavonoids production was the extremely low concentration of malonyl-CoA. An increased production of flavonoids was achieved by co-expressing acetyl-CoA carboxylase genes from *Photorhabdus luminescens* [44]. Also essential for flavonoid biosynthesis is the presence of UDP-glucose. Using the udg gene, researchers knocked out the endogenous system for consuming UDP-glucose resulting in an increase in intracellular UDP-glucose concentrations and subsequently increased flavanones and anthocyanins production [45].

Scientists were able to generate a wider range of natural and unnatural products when combining bacteria and eukaryotic cells in a pot. Using a modified *S. cerevisiae* strain, de novo generation of the important flavonoid intermediate naringenin from glucose was achieved for the first time, leading to four times higher concentrations than those seen in previous de novo biosynthesis experiments [46, 47].
