Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases

Abstract

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l -phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3′ prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo , the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3′-prenylnaringenin (3′-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.

Prenylation: A Critical Step for Biomanufacturing of Prenylated Aromatic Natural Products

“…In the first attempt, SfN8DT-1 was expressed in yeast to successfully synthesize 8-prenylnaringenin (8-PN) by feeding the substrate naringenin, 119 while the de novo synthesis of 8-PN in S. cerevisiae was achieved by introducing SfFPT in the subsequent research. 166 Beyond the plant aPTs, the sPT-type AnaPT from Neosartorya fischeri was first proven to catalyze naringenin at the 3′-position in yeast, and the titer of 3-prenylnaringenin (3-PN) is 1.10 μg/ L. 171 Later, the biosynthesis of 6-prenylnaringenin (6-PN) was achieved by ShFPT, a sPT-type aPTs from Streptomyces sp. NT11, in E. coli, and the titer of 6-PN was up to 69.9 mg/L with the DMAPP supply enhancement.…”

Plant Flavonoid Production in Bacteria and Yeasts

“…Expression of this enzyme in S. cerevisiae resulted in the bioconversion of naringenin to 8-prenylnaringenin (Sasaki et al, 2009). Moreover, SfN8DT expression in naringenin-producing S. cerevisiae resulted in the synthesis of 8-prenylnatingenin from glucose (Levisson et al, 2019), phenylalanine (Isogai et al, 2021) and p-coumaric acid (Li et al, 2015).…”

“…However, only the prenyltransferase SfN8DT-1 was identified, and enzymes responsible for the C6-and C3′-prenylation of naringenin remain unidentified. Recently, the in vivo production of 3′prenylnaringenin was achieved by the expression of fungal prenyltransferase with broad substrate specificity (Isogai et al, 2021). Such microbial promiscuous enzyme may also be able to catalyze prenylation of other positions of flavonoids, for which specific enzymes have not yet been identified.…”

Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

“…It is important to mention that as episomal plasmids were used as expression vectors, different yeast transformed colonies can have huge differences in terms of plasmid copy number and, consequently, in product concentration. Hence, future integration in the genome, as single or multiple gene copies, could result in more reproducible effects [57].…”