Engineering the expression of plant secondary metabolites-genistein and scutellarin through an efficient transient production platform in Nicotiana benthamiana L

Abstract

Plant natural products (PNPs) are active substances indispensable to human health with a wide range of medical and commercial applications. However, excessive population growth, overexploitation of natural resources, and expensive total chemical synthesis have led to recurrent supply shortages. Despite the fact that the microbial production platform solved these challenges, the platform still has drawbacks such as environmental pollution, high costs, and non-green production. In this study, an efficient platform for the production of PNPs based on the transient expression system of Nicotiana benthamiana L. combined with synthetic biology strategies was developed. Subsequently, the feasibility of the platform was verified by a simple "test unit." This platform was used to synthesize two high-value PNPs: genistein (5.51 nmol g^-1 FW) and scutellarin (11.35 nmol g^-1 FW). Importantly, this is the first report on the synthesis of scutellarin in heterologous plants. The platform presented here will possibly be adopted for the heterologous production of genistein and scutellarin in tobacco plants as a novel and sustainable production strategy.

Keywords: Nicotiana benthamiana; genistein; plant natural products; scutellarin; synthetic biology; transient production platform.

FIGURE 1

A schematic illustrating the assembly of multigene constructs based on the GGC. (A) Recognition sequence, cleavage site, and post-cleavage overhangs of two type IIS restriction enzymes (BsaI and BpiI). (B) The BsaI recognition sites (GGTCTC) and specific fusion sites (e.g., GGAG and ACAA) on both sides of each Level-0 module (e.g., promoter, UTR, ORF, 2A, and terminator) were introduced by primers. (C) Level-1 module digestion-ligation process. Each expression cassette was assembled into Level-1 destination vectors. (D) The final Level-2 modules construction process. Multiple Level-1 modules formed the final vector via a one-step digestion-ligation reaction of BpiI and T4 ligase. (E) Schematic diagram of T-DNA regions containing pathway genes involved in the synthesis of genistein and scutellarin. Part A, genistein; Part B, scutellarin; NPTII, aminoglycoside phosphotransferase from Tn5; Ω, tobacco mosaic virus 5′-leader sequence. 2A, Thosea asigna virus 2A.

FIGURE 2

A schematic diagram of the novel E-platform. (2–1) The structure of the transparent cube-mold. (2–2) Vacuum infiltration process. (A) The fabrication process of N. benthamiana. (B) The union of several N. benthamiana. (C) The leaves were inverted into a stainless steel-tank filled with A. tumefaciens suspension. Then, the tank was transferred to the vacuum infiltration device. (D) The infiltrated leaves contain two high-value PNPs.

FIGURE 3

Pre-test of the novel E-platform. (A) Schematic diagram of the simple “test unit.” (B) GFP fluorescence of the leaves under blue excitation light (approx. 470 nm). (C) Phenotypes of WT and infiltrated N. benthamiana. (D,E) Color reaction and total flavonoid content determination in WT and infiltrated N. benthamiana. “Transient” represents infiltrated N. benthamiana. The error bars represent the standard deviations from three independent experiments. The asterisks indicate significant differences (*P < 0.05).

FIGURE 4

Schematic overview of the genistein and scutellarin biosynthetic pathways in N. benthamiana. A simplified representation of flavonoids biosynthesis in N. benthamiana shows key intermediates (black) and enzymes (red). Naringenin (black box), as a crucial node, enters different metabolic pathways catalyzed by different heterologous enzymes. The enzymes in pink box (isoflavones) and blue box (flavones) are derived from soybean and E. breviscapus, respectively. The red arrow represents partial genes that can be activated by AtMYB12. PAL, phenylalanine ammonia lyase; CoA, coenzyme A; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; IFS, isoflavone synthase; HID, hydroxyisoflavanone dehydratase; F3H, flavanone-3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; RT, rhamnosyl transferase; 3GT, flavonol-3-glucosyltransferase; FLS, flavonol synthase; apigenin-7-O-G, apigenin-7-O-glucuronide; FNSII, flavone synthase II; UDPGDH, UDP-glucose dehydrogenase; F7GAT, flavonoid-7-O-glucuronosyltransferase; F6H, flavone-6-hydroxylase.

FIGURE 5

UPLC-MS analysis of genistein in the leaf extracts from infiltrated N. benthamiana (Detection wavelength, 254 nm). The EIC (m/z = 269.0460) of the genistein in the standard sample (A), WT (B), and infiltrated N. benthamiana (C). MS fragmentation patterns of (-)-genistein in the leaves of infiltrated N. benthamiana (E), and it was identical to the genistein standard (D).

FIGURE 6

Heterologous production of scutellarin in N. benthamiana tissues. EIC (m/z = 461.0727) of scutellarin in standard (A), WT (B), and infiltrated N. benthamiana tissues (C). MS fragmentation patterns of (-)-scutellarin standard (D). MS fragmentation patterns of (-)-scutellarin in N. benthamiana infiltrated tissues (E).