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Biocatalytic access to diverse prenylflavonoids by combining a regiospecific C-prenyltransferase and a stereospecific chalcone isomerase

Prenylflavonoids are valuable natural products that have diverse biological properties, and are usually generated biologically by multiple metabolic enzymes in nature. In this study, structurally diverse prenylflavonoids were conveniently synthesized by enzymatic catalysis by combining GuILDT, a regiospecific chalcone prenyltransferase, and GuCHI, a stereospecific chalcone isomerase that has promiscuous activity for both chalcones and prenylchalcones as substrates. Our findings provided a new approach for the synthesis of natural/unnatural bioactive prenylflavonoids, including prenylchalcones and optical prenylflavanones with chalcone origins.


Introduction
Prenylflavonoids are a diverse class of naturally occurring flavonoids that contain prenyl substituents. In higher plants, these compounds usually exhibit strong antibacterial and antifungal activities 1 . Moreover, many of them have attracted attention due to their diverse pharmacological activities, such as anti-cancer, anti-obesity, anti-inflammation activity, anti-bacterial pathogens, and the impairment of autophagosome maturation [2][3][4][5][6][7][8] . C-Prenylation of flavonoids by flavonoid prenyltransferase (PTase) represents a Friedel-Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylflavonoids and plays a crucial role in generating the diverse compounds due to the different prenylation positions and various types of prenyl groups as well as further tailoring modifications (e.g., hydroxylation and cyclization), leading to the production of more than 1000 prenylflavonoids in plants 9 . Substitutions with prenyls of different modes on the flavonoid scaffold not only contribute significantly to the structural diversity, but also markedly enhance the bioactivity and bioavailability compared with their nonprenylated parent molecules 10 .
The synthesis of prenylflavonoids by chemical methods has always had many limitations, such as regio-/stereoselectivity and variations in the number of prenyl groups attached to various flavonoid skeletons. In contrast, enzymatic catalysis is effective and superior in terms of regioselectivity and stereoselectivity in the directional synthesis of prenylflavonoids. However, only a few flavonoid PTases from plants have been identified to date, and most of them are strictly substrate-specific and regiospecific [11][12][13][14][15][16][17] . It has been demonstrated that most of the enzymes involved in flavonoid scaffold biosynthesis, including chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), isoflavone synthase (IFS), and flavanone 3β-hydroxylase (F3H) have broad substrate specificity 18,19 . Of these enzymes, CHI catalyzes the stereospecific isomerization of a chalcone into the corresponding (-)-flavanone via intramolecular cyclization with clear substrate flexibility 20,21 . To develop an effective method to synthesize prenylflavonoids, we focused on cascade reactions by combining chalcone PTase and CHI to produce prenylflavonoids with diverse structures (Scheme 1).
Licorice from the roots and rhizomes of various species of Glycyrrhiza (Leguminosae) is one of the most frequently used traditional medicines in China and in other countries 22,23 . More than 200 prenylated flavonoids have been identified in licorice, including prenylchalcones, prenylflavanones, prenylflavones, and prenylisoflavonoids 24,25 . Therefore, Glycyrrhiza is considered an ideal source for the discovery of biosynthetic enzymes/genes for the biosynthesis of prenylflavonoids. In this article, a new chalcone-specific PTase, GuILDT, and a new stereo-and regiospecific type II CHI, GuCHI, were characterized from Glycyrrhiza uralensis and employed for cascade reactions to produce diverse bioactive prenylflavonoids. Their enzymatic synthesis applications as well as roles they played in the biosynthesis of structurally different prenylflavonoids were also discussed.

1
H and 13 C NMR spectra were recorded on VNOVA SYSTEM-600 spectrometers using CD 3 COCD 3 or CD 3 OD as solvent. Optical rotations were determined using a Perkin-Elmer Model 341 LC polarimeter (Perkin-Elmer). Enzymatic products were analyzed with a 1200 HPLC system (Agilent) coupled with an LCQ Fleet ion trap mass spectrometer (Thermo Scientific). HPLC analyses were conducted with an RP-18 end-capped Purospher STAR LP column (250 mm × 4.6 mm, 5 μm, Merck Millipore) with a flow rate of 1 mL/min. UV detection was set at 290 or 380 nm, and the column was operated at 30°C. The solvent system consisted of a linear gradient (45%-95%, v/v) of methanol in water Scheme 1 Biocatalytic access to diverse prenylflavonoids by combining various biosynthetic enzymes. with formic acid (0.1%) over 20 min, followed by isocratic elution with methanol (100%) for 10 min. For LC-MS analyses, the LC effluent was introduced into an ESI source (post column splitting ratio, 2:1). Ultra-high purity helium was used as the collision gas, and high purity nitrogen was used as the nebulizing gas. The optimized parameters in the positive-and negative-ion modes were the following: sheath gas flow rate, 20 arbitrary units; auxiliary gas flow rate, 5 arbitrary units; spray voltage, 5.0 kV; capillary temperature, 350°C; source collision induced decomposition (CID), 35 V; tube lens offset voltage, −75 V. For full-scan MS analysis, the spectra were recorded in the range m/z 100-900. For the isolation of enzymatic products, semi-preparative RP-HPLC was performed with a YMC-Pack ODS-A column (250 mm × 10 mm, 5 μm, YMC, Kyoto, Japan) at a flow rate of 3 mL/min with a linear gradient (40-80%, v/v) of acetonitrile in water over 15 min. The chiral analysis of flavanone enantimoers were operated utilizing a Chiralpak AD-H column (250 mm × 4.6 mm, 5 μm, Daicel Chemical Industries, Toyoko, Japan). The mobile phase consisted of n-hexane and 2-propanol (85:15, v/v), and analysed at 30°C at a flow rate of 1.0 mL/min with detection at 290 nm.

cDNAs cloning of GuILDT
Total RNA was prepared from ten-day-old cultured cells of G. uralensis treated with methyl jasmonate (MJ, final concentration 0.1 mmol/L) using an E.Z.N.A. Plant RNA Kit (Omega Bio-Tek, Norcross, GA). First-strand cDNA was synthesized from 10 μg of total RNA in 20 μL with SmartScribe reverse transcriptase (Clontech, Mountain View, CA, USA).

Heterologous expression of GuILDT in Sacharomyces cerevisiae
The yeast expression vector pESC and pEASY-GuILDT were digested by EcoR I and Not I, and then GuILDT and digested vector were ligated overnight at 16°C with T4 ligase (NEB). The resulting plasmid, pESC-GuILDT was transformed into S. cerevisiae strain YPH499 by the modified LiCl method 29 .
For the expression of recombinant protein, the transformant YPH499-pESC-GuILDT were cultured overnight at 30°C in selective liquid media containing glucose as the carbon source. Cultures were collected by centrifugation, washed in sterile water, and re-suspended to an OD 600 ¼ 1.0 in fresh SG-His media containing galactose. After 24 h, the cells were harvested, resuspended in extraction buffer (Tris-HCl 100 mmol/L, pH 9.0 containing phenylmethylsulfonylfluoride (PMSF, 1.0 mmol/L)) and homogenized with acid-washed glass beads eight times in a vortex mixer (each cycles: 30 s vortexing and 30 s cooling on ice). The homogenized mixture was centrifuged (10,000× g, 20 min), then the supernatant was ultracentrifuged (160,000× g, 90 min, 4°C) to pellet the microsomal fraction. After washing, the membrane fraction was re-suspended with 100 mmol/L Tris-HCl buffer (pH 9.0), which was used as an enzyme solution for prenyltransferase assay described below. The total protein concentration was determined with the Bradford method using BSA as a standard 30 .

Heterologous expression in Escherichia coli and protein purification of GuCHI
GuCHI was inserted into the pET-28a bacterial expression vector at the Nde I and Sal I sites. His 6 -GuCHI was heterologously expressed in Rosetta (DE3) E. coli cells (Novagen, Madison, WI, USA). Bacterial cultures were grown in LB media containing 34 mg/L chloromycetin and 50 mg/L kanamycin overnight at 37°C . The cultures were diluted and then grown to an OD 600 0.4−0.6. At this point, expression of the recombinant protein was induced by addition of 0.1 mmol/L isopropyl-thio-β-D-galactoside (IPTG). After incubated overnight at 16°C, cultures were harvested by centrifugation at 4000× g for 10 min and washed with sterile water. After washing, collected cultures was frozen with liquid nitrogen and stored at −80°C.

Functional assay of GuCHI in vitro
Chalcones (1−4) and their prenylated derivatives (1a−4a) (40 mmol/L) were incubated with 200 μL of reaction buffer at 25°C for 5 min, extracted with ethyl acetate, and analyzed by HPLC, respectively. For prepared the cyclized products, chalcones and their prenylated derivatives were added into the reaction buffer containing purified His 6 -GuCHI protein. After incubation at 25°C for 15 min, the reaction mixtures were extracted with ethyl acetate. The extracts were subjected to semi-preparative reversed HPLC chromatography, respectively.
For kinetic properties study, the decrease of substrate A395 was monitor using a nano detector. Spectrophotometric operations were performed at 25°C in 0.5 mL reaction buffer. Reactions were initiated by the addition of enzyme, and initial rates were calculated based upon decrease of substrate A395. Steady-state kinetic parameters (K m ) were calculated by fitting the untransformed initial velocity data.

Results and discussion
In an effort to find the chalcone-specific PTase(s) gene in Glycyrrhiza, homology-based RT-PCR/RACE was performed 12,16,17 . One candidate PTases cDNA was isolated from the cultured cells of G. uralensis and encoded a polypeptide of 408 amino acids. The polypeptide was clustered into plant flavonoid PTases phylogenic clade and shared 53% identity with the previously reported plant flavonoid PTases (Fig. 1). Two conserved aspartate-rich motifs of plant PTases including NQXXDXXXD and KD(I/L)XDX(E/D)GD were observed, as well as nine putative transmembrane regions (Supporting information Fig. S1).
To investigate its prenylation activity with chalcone, the putative protein was heterologously expressed in the yeast YPH499. A microsomal fraction was prepared and incubated with isoliquiritigenin (1, molecular weight (MW) 256) and dimethylallyl diphosphate (DMAPP) in the presence of MgCl 2 . The enzymatic reaction (100 mmol/L Tris-HCl, pH 9.0; 0.2 mmol/L 1; 0.4 mmol/L DMAPP; 400 μg of microsomes containing recombinant protein; 30°C, 12 h) was analyzed by HPLC-ESI/MS, which gave an ion peak [M -H] − at m/z 323, with 68 amu larger MW than that of 1, indicating the formation of a mono-prenylated product (Fig. 2). The product (1a) was further prepared in a scaled-up reaction and identified by NMR. A comparison of the 1 H NMR spectra of 1a and 1 revealed that one dimethylallyl moiety had been introduced and that one methylene proton signal at δ H 3.35 (d, J ¼ 7.2 Hz, H 2 -1″), one olefinic proton signal at δ H 5.27 (t, J ¼ 7.2 Hz, H-2″), and two methyl signals at δ H 1.78 and 1.64 (H 3 -4″ and H 3 -5″) had appeared. The disappearance of H-3′ of 1 suggested the dimethylallyl moiety was attached at the C-3′ position. In addition, the downfield shift of C-3′ from δ C 99.8 in 1 to δ C 108.0 in 1a further confirmed that deduction. Thus, the structure of 1a was unambiguously determined as isobavachalcone 44 , and the enzyme was designated GuILDT (G. uralensis isoliquiritigenin dimethylallyltransferase, GenBank accession code KR139751).
Furthermore, great effort was made to identify a prenylchalconerecognized CHI from the cell cultures of G. uralensis. In nature, CHI catalyzes the stereo-specific isomerization of chalcones into the corresponding (−)-flavanones as intermediates of subsequent flavonoid biosynthesis [45][46][47] . However, this reaction can occur chemically and yields two enantiomers, (þ)-and (−)-flavanones. A homology-based PCR/RACE strategy was used to obtain a full-length CHI cDNA from cultured G. uralensis cells based on two conserved amino acid sequences, GXFXKFT and (M/I)IGXH(G/A). This sequence contained an open reading frame (690 bp) encoding a polypeptide of 229 amino acids that showed a 43%-53% identity with various type I CHIs and 74%-80% with type II CHIs (Supporting information Fig. S6). It was clustered into the latter group and designated as GuCHI (G. uralensis chalcone isomerase, GenBank accession code KR139752, Fig. 3).
The recombinant GuCHI was heterologously expressed in E. coli as a His 6 -tagged protein and subsequently purified by affinity chromatography for enzymatic assays. The incubation of 1 with GuCHI (50 mmol/L Tris-HCl, 50 mmol/L NaCl, 1 mmol/L DTT, 1% glycerol, pH 7.5, 25°C, 5 min) yielded one product, which was identified as (−)-liquiritigenin (1b) by NMR and CD spectroscopy. Additionally, GuCHI can also catalyze the reaction of other chalcones (2−4) to the corresponding flavanones (Fig. 4, see Supporting information for details). It is noteworthy that 2 can be spontaneously isomerized into (7)-naringenin in aqueous solution. A chiral HPLC analysis of the products gave (−)-naringenin in 90% yield, indicating the predominant contribution of enzymatic cyclization by GuCHI in this process (Supporting information Fig. S7A).
were observed and prepared, and their structures were identified as the corresponding (−)-prenylflavanones by MS, 1 H NMR, and 13 C NMR spectroscopy (Fig. 4, see Supporting information for details). Both (−)-8-prenylnaringenin ((−)-2c-A, in 85% yield with 98% ee value) and (−)-6-prenylnaringenin((−)-2c-B, in 15% yield with 56% ee value) were generated when 2a was reacted with GuCHI, which indicated that the isomerization by GuCHI is stereoand regio-specific (Supporting information Fig. S7B-C). Notably, enzymatic catalysis played a vital role in the regio-specific cyclization of levorotatory flavanone, although spontaneous cyclization was also observed. GuCHI exhibited substrate flexibility and accepted a broad range of chalcones with either different hydroxyl substitution patterns in ring A or C-3′/C-5′ prenylation in ring A′. However, the cyclization of prenylated chalcone gave much slower kinetics than did a non-prenylated chalcone. For example, the K m values of 1 and 2 were 15 and 3 μmol/L, respectively, and that of 1a and 2a were 32 and 11 μmol/L, respectively (Supporting information Fig. S8).
In summary, in G. uralensis, we characterized GuILDT, a new membrane-bound chalcone-specific PTase with C-3′ regio-specificity, and GuCHI, a new stereo-and regio-specific type II CHI with substrate promiscuity. GuCHI is the first reported CHI that catalyzes the intramolecular cyclization of prenylchalcones to generate prenylflavanones. The results reported herein provide a new approach to the enzymatic synthesis of prenylflavonoids and create the possibility of producing diverse bioactive natural/unnatural prenylflavonoids by enzymatic synthesis, even by combinatorial biocatalysis with other enzymes. In addition, considering the checkpoint role of chalcones (naringenin chalcone and isoliquiritigenin) in the flavonoid biosynthetic pathway and the promiscuity of the tailoring enzymes [45][46][47] , the results suggest that the prenyl moiety might also been introduced at the chalcone stage to yield prenylchalcones, which subsequently act as common intermediates involved in the biosynthesis of a variety of structurally different prenylflavonoids.