Biochemical Characterization of a Novel Prenyltransferase from Streptomyces sp. NT11 and Development of a Recombinant Strain for the Production of 6-Prenylnaringenin

Abstract

Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (ShFPT) gene from Streptomyces sp. NT11 was expressed in Escherichia coli, and its biochemical characteristics were determined. ShFPT exhibited high selectivity to prenylate naringenin at C-6 to generate 6-prenylnaringenin. The optimal activity was observed at pH 6.0 and 55 °C. The K cat and K m for naringenin were 0.0095 s −1 and 0.20 mM, respectively. Several promiscuous kinase and isopentenyl phosphate kinase genes were screened to develop the most efficient dimethylallyl diphosphate (DMAPP) synthesis pathway for 6-prenylnaringenin synthesis in E. coli. The 6-prenylnaringenin production was improved by changing the induction strategies and optimizing the bioconversion conditions. Finally, 6-prenylnaringenin production reached the highest yield of 69.9 mg/L with average productivity of 4.0 mg/L/h after 16 h incubation, which is the highest yield for any prenylated flavonoid reported to date in E. coli. Therefore, this study provides an efficient method for 6-prenylnaringenin production and reveals the DMAPP synthesis pathway.

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade

“…The highest production of DMAPP reached 1.21 mM with the addition of 80 mM DMA ( Figure 12 B). Similar results have been reported with the recombinant strains, and the highest yields were observed using 20~25 mM prenol in vivo [ 20 , 39 ]. The half-saturation constant of DMA for the one-pot enzymatic cascade was ~12 mM—significantly higher than the Km values of SfPK and MtIPK.…”

“…Isopentenyl phosphate kinases from M. tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and A. thaliana (AtIPK) have been used to reconstruct the IUP in the recombinant strains for improvement of the supply of DMAPP [ 4 , 29 , 36 , 37 , 38 , 39 ]. Thus, in this study, MtIPK, MthIPK, and AtIPK were chosen to characterize the biochemical properties.…”

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

“…NT11, in E. coli, and the titer of 6-PN was up to 69.9 mg/L with the DMAPP supply enhancement. 69 Moreover, Wang et al designed an artificial icaritin biosynthetic pathway by identifying a novel aPT from Epimedium sagittatum (EsPT2), which catalyzed the prenylation of kaempferol at the C-8 position, and the icaritin titer reached 19.7 mg/L by coculturing yeast and E. coil. 115 Although the accumulation of acceptor flavonoids and prenyl donors through some metabolic strategies has been well studied, 197−199 the titer of prenylated flavonoids in MCFs is still low so far, and the ratelimiting step is usually the prenylation mediated by flavonoid aPTs.…”

“…NT11, in E. coli, and the titer of 6-PN was up to 69.9 mg/L with the DMAPP supply enhancement . Moreover, Wang et al designed an artificial icaritin biosynthetic pathway by identifying a novel aPT from Epimedium sagittatum (EsPT2), which catalyzed the prenylation of kaempferol at the C-8 position, and the icaritin titer reached 19.7 mg/L by coculturing yeast and E.…”

Heterologous biosynthesis of prenylated resveratrol through multiplex metabolic engineering in Escherichia coli

“…Hence, microbial synthesis of these chemicals presents an attractive, green alternative due to lower ecological and energy costs as well as higher productivity. [15][16][17][18] The biosynthesis of resveratrol starts from L-phenylalanine or L-tyrosine in plants. Phenylalanine/tyrosine ammonia-lyase (PAL/TAL) catalyzes the deamination and the formation of p-coumaric acid ( p-CA), in which cinnamic 4-hydroxylase (C4H) is involved when L-phenylalanine is used as the substrate.…”