Tailored biosynthesis of diosmin through reconstitution of the flavonoid pathway in Nicotiana benthamiana

Abstract

The flavonoid diosmin (diosmetin 7-O-rutinoside) is used as a therapeutic agent for disorders of the blood vessels such as hemorrhoids and varicose veins. Diosmin is commercially produced using semi-synthetic methods involving the oxidation of hesperidin, the most abundant flavonoid in citrus fruits. However, this method produces byproducts that are toxic to the environment, and new sustainable methods to produce diosmin are required. Here, we used a synthetic biology approach to produce diosmin without generating toxic byproducts through reconstitution of the diosmin biosynthetic pathway in Nicotiana benthamiana. We first established that N. benthamiana leaves co-infiltrated with all seven genes in the flavonoid biosynthesis pathway produced high levels of luteolin, a precursor of diosmetin. We then compared the activity of modification enzymes such as methyltransferases, glucosyltransferases, and rhamnosyltransferases in Escherichia coli and in planta and selected genes encoding enzymes with the highest activity for producing diosmetin, diosmetin 7-O-glucoside, and diosmin, respectively. Finally, we reconstructed the entire diosmin biosynthetic pathway using three constructs containing ten genes encoding enzymes in this pathway, from phenylalanine ammonia lyase to rhamnosyltransferase. N. benthamiana leaves transiently co-expressing all these genes yielded 37.7 µg diosmin per gram fresh weight. To our knowledge, this is the first report of diosmin production in a heterologous plant system without the supply of a precursor. Successful production of diosmin in N. benthamiana opens new avenues for producing other commercially important flavonoids using similar platforms.

Keywords: Nicotiana benthamiana; diosmetin; diosmin; flavonoid; synthetic biology; transient expression.

Figure 1

Proposed diosmin biosynthetic pathway from phenylalanine. The phenylpropanoid pathway (blue box) starts with phenylalaine produced by the shikimate pathway. The diosmin (red box) biosynthesis pathway starts with chalcone synthase (CHS) converting three molecules of malonyl-CoA and one molecule of 4-coumaroyl-CoA into naringenin chalcone. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; F3'H, flavonoid 3'-hydroxylase; F4′OMT, flavonoid 4′-O-methyltransferase; F7GT, flavonoid 7-O-glucosyltransferase; 1,6RhaT, 1,6-rhamnosyltransferase.

Figure 2

Luteolin production in N. benthamiana leaves with differently reconstituted flavonoid biosynthesis modules. N. benthamiana leaves were agro-infiltrated with Agrobacterium tumefaciens strain GV3101 harboring the single gene expression cassettes P+C+F+F (P, AtPAL; C, OsCHS; F, OsFNS; F, OsF3′H) or the multigene expression cassettes PCFF (P, AtPAL; C, OsCHS; F, OsFNS; F, OsF3′H), or PC4 (P, AtPAL; C, AtC4H; 4, Sh4CL) and CCFF (C, OsCHS; C, BrCHI; F, OsFNS; F, OsF3′H) in binary vectors. GV3101 (GV) was used as a negative control. UPLC-DAD-QToF/MS analysis of extracts from leaves was conducted in positive ion mode. (A) Schematic diagram of single (P+C+F+F) or multigene (PCFF, PC4, CCFF) expression vectors of target genes. (B) Extracted-ion chromatogram (XIC) at m/z 287.05 representing protonated luteolin aglycone in infiltrated leaves. (C) Representative XICs showing mass spectra of the luteolin standard and products detected in infiltrated leaves. (D) Flavonoid contents in the three independent biological samples were calculated based on the area of standards (naringenin, eriodictyol, apigenin, luteolin). The mean values ± SE (standard error) of three independent biological samples are shown.

Figure 3

Screening of an optimum F4′OMT for diosmetin production. (A) Diosmetin is produced from luteolin by flavonoid-4′-O-methyltransferase (F4′OMT). (B) Expression of recombinant F4′OMT enzymes in E. coli Rosetta (DE3). Proteins were separated on a 10% SDS-polyacrylamide gel. V.C, vector control. (C) Substrate-feeding assays of recombinant flavonoid 4′-O-methyltransferases (F4′OMTs) using luteolin as the substrate. PaF4′OMT (Plagiochasma appendiculatum); MpOMT (Mentha × piperita); SOMT2 (Glycine max); CreOMT1 (Citrus reticulata); CreOMT4 (C. reticulata). (D) UPLC analysis of diosmetin standard and flavonoid extracts from leaves infiltrated with mixtures of Agrobacterium GV3101 strains harboring binary vectors containing multigene expression cassettes [PC4+CCFF: PC4 (P, AtPAL; C, AtC4H; 4, Sh4CL); CCFF (C, OsCHS; C, BrCHI; F, OsFNS; F, OsF3′H)] and a single (PaF4′OMT) gene expression cassette. GV3101 (GV) was used as a negative control. (E) Flavonoid aglycone profiles in the infiltrated leaves were analyzed by UPLC-DAD-QToF/MS in positive ion mode. Extracted-ion chromatogram (XIC) at m/z 301.07 of protonated diosmetin aglycone in the infiltrated leaves. (F) Representative XICs showing mass spectra of diosmetin standard and products in the infiltrated leaves. (G) Flavonoid contents in three independent biological samples were calculated based on the area of corresponding standards (naringenin, eriodictyol, apigenin, luteolin, and diosmetin). Mean values ± SE (standard error) of three independent biological samples are shown.

Figure 4

Impact of co-expressing glucosyltransferase with the diosmetin production module in N. benthamiana. N. benthamiana leaves were agro-infiltrated with Agrobacterium tumefaciens GV3101 strains harboring binary vectors containing multigene expression cassettes [PC4+CCFF: PC4 (P, AtPAL; C, AtC4H; 4, Sh4CL); CCFF (C, OsCHS; C, BrCHI; F, OsFNS; F OsF3′H)] and single gene expression cassettes [PaF4′OMT, CsUGT76F1]. Flavonoid profiles in the infiltrated leaves were analyzed by UPLC-DAD-QToF/MS in positive ion mode. (A) Diosmetin 7-O-glucoside is produced from diosmetin by flavonoid 7-O-glucosyltransferase (F7GT). (B) XICs at m/z 463.12 representing protonated diosmetin-7-O-glucoside in N. benthamiana leaves agro-infiltrated with a GV3101 strain harboring binary vectors containing multiple genes (PC4+CCFF, CCFF) and single gene (PaF4′OMT, F7GT). (C) Representative XICs showing mass spectra of diosmetin-7-O-glucoside standard and products in the infiltrated leaves. (D) Diosmetin-7-O-glucoside contents in three independent biological samples were calculated based on the area of the standards. Mean values ± SE (standard error) of three independent biological samples are shown.

Figure 5

Diosmin production in N. benthamiana leaves by multigene expression and co-expression of target genes. Flavonoid profiles in E. coli were analyzed by UPLC-DAD-QToF/MS in negative ion mode. (A) Diosmin is produced by 1,6-rhamnosyltransferase (1,6RhaT) from diosmetin 7-O-glucoside. Substrate-feeding assays of recombinant 1,6-rhamnosyltranferase (1,6RhaT). (B) Proteins were separated on a 10% SDS-polyacrylamide gel. V.C, vector control. Cs1,6RhaT (Citrus sinensis), CiRhaT-GD4x (GD4x, Chrysanthemum indicum), CiRhaT-AH2x/CiRhaT-HB2x (AH2x, Chrysanthemum indicum). (C) Extracted-ion chromatograms (XICs) at m/z 461.11 and m/z 607.16 representing deprotonated diosmetin 7-O-glucoside and diosmin in E. coli Rosetta (DE3). (D) Representative XICs showing mass spectra of diosmin standard and products extracted from E. coli expressing recombinant RhaT enzymes. (E) Flavonoid aglycones (apigenin, luteolin, diosmetin) contents in N. benthamiana leaves agro-infiltrated with GV3101 strains harboring binary vectors containing multiple genes [PC4+CCFF: PC4 (P, AtPAL; C, AtC4H; 4, Sh4CL); CCFF (C, OsCHS; C, BrCHI; F, OsFNS; F, OsF3′H)] and single genes [PaF4′OMT, CsUGT76F1, Cs1,6RhaT]. (F) Flavonoid glycosides (diosmetion-7-O-glucoside and diosmin) contents in N. benthamiana leaves agro-infiltrated with GV3101 strains harboring binary vectors containing multiple genes (PC4+CCFF) and single genes (PaF4′OMT, CsUGT76F1, Cs1,6RhaT). Average flavonoid aglycone and glycoside contents were calculated based on the areas of the corresponding standards. Mean values ± SE (standard error) of three independent biological samples are shown.

Figure 6

Diosmin production in N. benthamiana by transient co-expression of three modules reconstituting the diosmin biosynthesis pathway. N. benthamiana leaves were agro-infiltrated with GV3101 strains harboring binary vectors containing three modules: PC4 (P, AtPAL; C, AtC4H; 4, Sh4CL), CCFF (C, OsCHS; C, BrCHI; F, OsF3′H; F, OsFNS), and MGR (M, PaF4′OMT; G, CsUGT76F1; R, Cs1,6RhaT). Flavonoid glycoside profiles in the infiltrated leaves were analyzed by UPLC-DAD-QToF/MS in positive ion mode. (A) Schematic diagram of multigene expression vectors for target genes; module 1, PC4 (P, AtPAL; C, AtC4H; 4, Sh4CL); module 2, CCFF (C, OsCHS; C, BrCHI; F, OsF3′H; F, OsFNS); module 3, MGR (M, PaF4′OMT; G, CsUGT76F1; R, Cs1,6RhaT). (B) Extracted-ion chromatogram (XIC) at m/z 609.18 representing protonated diosmin standard and products from the infiltrated leaves. (C) Representative XICs at m/z 609.18 showing MS/MS spectra of diosmin standard and products from the infiltrated leaves. (D) Flavonoids glycosides (diosmetin 7-O-glucoside, diosmin) contents in the agro-infiltrated leaves. Average diosmetin 7-O-glucoside and diosmin contents were calculated based on the areas of the corresponding standards. Mean values ± SE (standard error) of three independent biological samples are shown.