Plant Biotechnol. 37(3)

Plant Biotechnology 37, 301–310 (2020) DOI: 10.5511/plantbiotechnology.20.0421a

Original Paper

Molecular cloning and biochemical characterization of isoflav-3-ene synthase, a key enzyme of the biosyntheses of (+)-pisatin and coumestrol

Kai Uchida1, Toshio Aoki1,a, Hideyuki Suzuki2, Tomoyoshi Akashi1,* 1 Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan; 2 Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan * E-mail: [email protected] Tel & Fax: +81-466-84-3353

Received March 27, 2020; accepted April 21, 2020 (Edited by M. Mizutani)

Abstract Most leguminous plants produce (−)-type enantiomers of pterocarpans as the phytoalexin, but pea (Pisum sativum L.) produces the opposite stereoisomer of pterocarpan, (+)-pisatin. Biosynthesis of (−)-pterocarpan skeleton is completely characterized at the molecular level, and pterocarpan synthase (PTS), a dirigent (DIR) domain-containing protein, participates in the last dehydration reaction. Similarly, isoflav-3-ene, a precursor of (+)-pisatin, is likely to be biosynthesized by the DIR-mediated dehydration reaction; however the biosynthesis is still unknown. In the present study, we screened PTS homologs based on RNA-sequence data from (+)-pisatin-producing pea seedlings and demonstrated that one of the candidates encodes isoflav-3-ene synthase (I3S). Real-time PCR analysis revealed that transcripts of I3S, in addition to other genes involved in the (+)-pisatin pathway, transiently accumulated in pea upon elicitation prior to the maximum accumulation of (+)-pisatin. I3S orthologs were also found in soybean and Lotus japonicus that are not known to accumulate (+)-pterocarpan, and the catalytic function of gene products was verified to be I3S by the in vitro enzyme assay. Incubation of the crude extract of elicited soybean cells with isoflav-3-ene yielded coumestrol, suggesting that isoflav-3-ene is a precursor of coumestrol biosynthesis in soybean. Key words: coumestrol, isoflav-3-ene, phytoalexin, (+)-pisatin, Pisum sativum.

(−)-type enantiomers, and only a limited number Introduction of plant species, such as peanut (Arachis hypogaea), Phytoalexins are plant-producing antimicrobial Japanese pagoda tree (Styphnolobium japonicum), and compounds induced by both biotic and abiotic pea (Pisum sativum), produce (+)-pterocarpans (Ingham stresses, and leguminous plants mainly produce 1979; Strange et al. 1985; VanEtten et al. 1989). The pterocarpan-based isoflavonoids as phytoalexins (Aoki stereochemistry of pterocarpan is important because it et al. 2000; Ingham 1982). Pterocarpans contain two determines the antimicrobial activity against pathogens; asymmetric carbons at C-6a and C-11a, but only two that is, some plant pathogens can detoxify (−)-isomers cis configurations are sterically possible and found but not (+)-isomers, and as a result, (+)-pterocarpans in nature (Figure 1) (Dewick 1986). All levorotatory show higher activity than the (−)-isomers (Delserone et pterocarpans are widely accepted to have the (6aR,11aR) al. 1992). The entire biosynthesis of (−)-pterocarpan was configuration, such as (−)-medicarpin (6a) and revealed by identifying pterocarpan synthase (PTS) as (−)-maackiain (6b), and dextrorotatory pterocarpans the long-standing missing link (Uchida et al. 2017), but have the opposite configuration, such as (+)-maackiain further studies are required to elucidate the biosynthesis (7a) and (+)-pisatin (7c) (Slade et al. 2005). Among the of (+)-pterocarpan. two enantiomers of pterocarpans, most legumes produce (+)-Pisatin (7c), which was the first chemically

Abbreviations: DIR, dirigent; DMDI, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol; DMDIF, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene; DMI, 7,2′-Dihydroxy-4′-methoxyisoflavanol; DMIF, 7,2′-dihydroxy-4′-methoxyisoflav-3-ene; HPLC, high performance liquid chromatography; I2′H, isoflavone 2′-hydroxylase; I3S, isoflav-3-ene synthase; I4R, 2′-hydroxyisoflavanone 4-reductase; IFR, isoflavone reductase; LB, Luria Broth; NMR, nuclear magnetic resonance; PTS, pterocarpan synthase; RPKM, reads per kilobase of exon model per million mapped reads; RT-PCR, reverse transcription polymerase chain reaction; SOR, sophorol reductase; THIF, 7,2′,4′-trihydroxyisoflav-3-ene; TLC, thin-layer chromatography; VR, vestitone reductase. a Deceased This article can be found at http://www.jspcmb.jp/ Published online July 30, 2020

Figure 1. Biosynthesis of (+)-pisatin and related compounds. Names of skeletons and individual compounds are shown in bold and plain fonts, respectively. Isoflav-3-enes are shown inside the dotted-line frame. Biosynthesis of isoflavones and 2′-hydroxyisoflavones shown here constitutes a metabolic grid, and enzymes involved are shown in parentheses. Constituents of isoflavonoids of leguminous plants are as follows: (6a) Glycyrrhiza spp. and Medicago spp., (6b) Maackia spp., Cicer spp., and Trifollium spp., (7a) Styphnolobium japonicum, (7c) Pisum sativum, (8) Glycine max, Glycyrrhiza spp., Lotus japonicus, and Medicago spp., (9) Cicer spp., (10) Lespedeza homoloba, and (11) Vigna unguiculate. Abbreviations: IFS, 2-hydroxyisoflavanone synthase; HID, 2-hydroxyisoflavanone dehydratase; HI4′OMT, 2-hydroxyisoflavanone 4′-O- methyltransferase; I2′H, isoflavone 2′-hydroxylase; I3′H, isoflavone 3′-hydroxylase; I3S, isoflav-3-ene synthase; IFR, isoflavone reductase; I4R, 2′-hydroxyisoflavanone 4-reductase; PBS, pseudobaptigenin synthase; PTS, pterocarpan synthase; SOR, sophorol reductase; VR, vestitone reductase; 2′HF, 2′-hydroxyformononetin; DMD, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavone; DMI, 7,2′-dihydroxy-4′-methoxyisoflavanol; DMDI, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol; DMIF, 7,2′-dihydroxy-4′-methoxyisoflav-3-ene; DMDIF, 7,2′-dihydroxy-4′,5′- methylenedioxyisoflav-3-ene; THIF, 7,2′,4′-trihydroxyisoflav-3-ene.

identified phytoalexin and is exclusively produced by pea hydroxyisoflavanones (Fischer et al. 1990; Paiva et al. (Cruickshank and Perrin 1960), is one of the best-studied 1991; Tiemann et al. 1991; Uchida et al. 2017). An IFR (+)-pterocarpans but its biosynthesis has only been isolated from (+)-pisatin-producing tissue of pea did partially elucidated. The biosynthesis of (−)-pterocarpan not produce the expected (3S)-2′-hydroxyisoflavanone has been found to involve two stereospecific and yielded only (3R)-enantiomer (Paiva et al. 1994). intermediates, namely (3R)-2′-hydroxyisoflavanone The involvement of an epimerase, which converts (3R)- and (3R,4R)-2′-hydroxyisoflavanol, the latter of sophorol (3b) into (3S)-sophorol, has been previously which is converted to (−)-pterocarpan by PTS (Figure suggested (Dewick 1986; Paiva et al. 1994). However, in 1). Thus, clarifying the branching point to produce a tracer experiment, 3H-labeled (3R)-sophorol (3b) was opposite stereoisomers is crucial for the elucidation efficiently incorporated into+ ( )-pisatin (7c) compared of (+)-pterocarpan biosynthesis. As isoflavone with that from (3S)-sophorol (DiCenzo and VanEtten reductase (IFR) catalyzes the first introduction step of 2006), showing no evidence to support the hypothetical chirality in pterocarpan biosynthesis, it was originally epimerase. postulated to be the pivotal enzyme that determines the The next step to IFR is the conversion of (3R)-2′- stereochemistry of the end product (Banks and Dewick hydroxyisoflavanone to (3R,4R)-2′-hydroxyisoflavanol, 1982a, b). Indeed, several IFRs of the (−)-pterocarpan- and 2′-hydroxyisoflavanone 4-reductase (I4R) mediates producing legumes, such as soybean (Glycine max), this reaction. I4R is also designated as a sophorol alfalfa (Medicago sativa), and chickpea (Cicer arietinum), reductase (SOR) in pea (DiCenzo and VanEtten converted achiral 2′-hydroxyisoflavones into (3R)-2′- 2006) and vestitone reductase (VR) in alfalfa (Guo

Copyright © 2020 Japanese Society for Plant Biotechnology K. Uchida et al. 303 and Paiva 1995), and pea SOR specifically converts efficient synthesis of isoflav-3-enes from isoflavones (3R)-sophorol (3b) to (3R,4R)-7,2′-dihydroxy-4′,5′- was developed using co-cultured E. coli cells expressing methylenedioxyisoflavanol (DMDI, 4b). The suppressed several biosynthetic genes. Moreover, metabolic expression of the IFR and SOR genes by RNA-mediated correlation between the consumption of isoflav-3-ene genetic interference in the hairy roots of pea led and production of coumestrol was also observed in the to a decrease in the (+)-pisatin (7c) accumulation in vitro assay using crude extract of elicited soybean (Kaimoyo and VanEtten 2008). Taken together, these cells. The results achieved herein will offer a new results indicate that (+)-pisatin (7c) is biosynthesized perspective for the elucidation of stereoisomer-specific via (3R)-sophorol (3b) and (3R,4R)-DMDI (4b). (+)-pterocarpan biosynthesis, mediated by DIR domain- More recently, in vitro enzyme assays using a cell-free containing proteins that produce isoflav-3-ene. extract of (+)-pisatin-producing pea seedlings have demonstrated the conversion of (3R,4R)-DMDI (4b) Materials and methods to 7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene (DMDIF, 5b). Consequently, the achiral isoflav-3- Chemicals ene was proposed to be an intermediate of (+)-pisatin Formononetin (1a) and coumestrol (8) were purchased from biosynthesis (Celoy and VanEtten 2014). Thus, the Tokyo Chemical Industry (Tokyo, Japan) and Sigma-Aldrich molecular characterization of isoflav-3-ene synthase (St. Louis, MO, USA), respectively. (±)-Maackiain and (I3S, also termed 2′-hydroxyisoflavanol 3,4-dehydratase) (3R,4R)-DMDI (4b) were kindly donated by Dr. VanEtten is essential to clarify the biosynthetic pathway of HD (University of Arizona). DMDIF (5b) was prepared from (+)-pisatin (7c). maackiain according to a previous report (Martin and Dewick To date, numerous studies have revealed the various 1980). (3R,4R)-7,2′-Dihydroxy-4′-methoxyisoflavanol (DMI, biological activities of isoflav-3-enes. Based on chemical 4a) was obtained from laboratory stock (Uchida et al. 2017). ecology, judaicin (9) of Cicer plants has antifeedant (+)-Pisatin (7c) was isolated from elicited pea sprouts. activity against the herbivorous pest insect, Helicoverpa Pea sprouts purchased from a grocery store were soaked in armigera (Simmonds and Stevenson 2001). Furthermore, 10 mM CuCl2 for 1 h and rinsed with distilled water three times. phenoxodiol (haginin E, 7,4′-dihydroxyisoflav-3- After 24 h incubation at 25°C, leaves and stems were collected ene, 10) of Lespedeza homoloba induces apoptosis in and soaked in hexane. Subsequently, the hexane extracts were chemoresistant ovarian cancer cells, thereby exerting evaporated, and (+)-pisatin (7c) was purified by successive significant antitumor activity (Kamsteeg et al. 2003). In silica-gel thin-layer chromatography (TLC, Silica-gel F254, isoflavonoid biosynthesis, isoflav-3-enes are predicted Merck, Darmstadt, Germany) developed with the solvents, to be intermediates in the biosynthesis of coumestan toluene : ethyl acetate=3 : 1 (v/v) and toluene : ethyl acetate : (coumestrol, 8) and 2-arylbenzofuran (vignafuran, 11) methanol : light petroleum (6 : 4 : 1 : 3, v/v/v/v). The nuclear (Kinoshita 1997; Martin and Dewick 1980). Isoflav-3- magnetic resonance (NMR) spectra were recorded on an ECA- enes are labile compounds and their content in plant cells 500 system (JEOL, Tokyo, Japan). is very low; therefore, the identification and application of I3S would facilitate further studies on the biological Plant materials and elicitation activities of isoflav-3-ene-related isoflavonoids. Elicitation of pea seedlings was carried out according to Recently, we biochemically identified PTS proteins a previous report with slight modifications (DiCenzo and of three leguminous plants and found that they belong VanEtten 2006). Briefly, surface-sterilized pea seeds (Pisum to the b/d subfamily (DIR-b/d) of the dirigent (DIR) sativum cv. Usui, Takii Seed, Kyoto, Japan) were germinated on domain-containing protein (Uchida et al. 2017). They moist vermiculite and grown for 6 days in the dark. Cotyledons form a clade (PTS clade) with their orthologues with excised from seedlings were placed on filter paper soaked which they share >75% amino acid sequence identity. with 5 mM CuCl2 and periodically harvested. For the control, We also showed that a considerable number of PTS-like cotyledons were soaked with sterile distilled water. Elicited and proteins from leguminous plants form a monophyletic control pea cotyledons were extracted with methanol, and the group within a leguminous DIR-like clade. As I3S extracts were analyzed by HPLC using the conditions defined shares the substrate with PTS and catalyzes a similar as ‘program 1’ (Supplementary Data S1). The concentration of dehydration reaction, suggesting structural similarity of (+)-pisatin (7c) was calculated from the area under the curve I3S with the PTS clade. In the present study, we screened of the peak of the compound. the RNA-sequence data of (+)-pisatin-producing pea seedlings for DIR protein-like transcripts and RNA-sequence and de novo assembly demonstrated that one of the candidates encodes I3S. Total RNAs were isolated from elicited (10 mM CuCl2 for Its orthologues from soybean and Lotus japonicus were 24 h) pea seedlings using the SV Total RNA Isolation System also identified by homology-dependent gene isolation (Promega, Madison, WI, USA). RNA quality was evaluated on and biochemical characterization. Furthermore, an an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto,

CA, USA). The cDNA library was prepared using the TruSeq 12 ng) for 10 min. Reaction mixtures were extracted with ethyl RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) acetate and analyzed by HPLC using the conditions defined as and subjected to sequencing on the Illumina Miseq platform ‘program 2’ (Supplementary Data S1). with 150 bp paired-end reads. Reads were de novo assembled with CLC Genomics Workbench ver 5.5 (CLC bio, Aarhus, Phylogenetic analysis Denmark) to obtain 47,799 contigs. Phylogenetic analysis was performed using MEGA6 software (Tamura et al. 2013). Amino acid sequences were aligned using cDNA cloning and vector construction ClustalW, and the phylogenetic tree was constructed with cDNAs were synthesized from the total RNAs of elicited pea the default settings of neighbor-joining method with 1,000 cotyledon using a SuperScript III First Strand Synthesis bootstrap replicates. System for RT-PCR (Invitrogen, Carlsbad, CA, USA). The coding sequences of PsIFR (DDBJ accession no. AAB31368) Real-time PCR analysis and PsSOR (DDBJ accession no. AF107404), and N-terminus Total RNAs were isolated as described above, and cDNAs were truncated PsI3S1, LjI3S1, LjI3S2, and GmI3S1 were amplified synthesized using ReverTra Ace qPCR RT Master Mix with and cloned into the vectors (pCR8/GW/TOPO for PsIFR, gDNA Remover (Toyobo, Osaka, Japan). Real-time PCR was PsSOR, and PsI3S1; pET46 Ek/Lic for LjI3S1, LjI3S2, and carried out as described previously (Uchida et al. 2017) using GmI3S1) using PrimeSTAR HS DNA polymerase (TaKaRa, primer sets (Supplementary Data S2). Shiga, Japan), the primer sets (Supplementary Data S2), and the cDNA templates prepared from elicited pea cotyledons or L. One-pot synthesis of isoflav-3-ene from isoflavone japonicus and soybean cDNAs (Uchida et al. 2015). A multiple Recombinant E. coli C41 (DE3) cells harboring LjCPR1 cDNA expression vector was constructed using the In-Fusion cloning and codon-optimized LjI2′H gene (Uchida et al. 2015) or system (TaKaRa). Briefly, coding regions of PsIFR, PsSOR, and harboring PsIFR, PsSOR, and PsI3S1 cDNAs were pre-cultured N-terminus truncated PsI3S1 cDNAs were transferred into the separately in 50 ml of LB liquid medium containing 50 mg l−l pET-53-DEST vector using a Gateway System (Invitrogen). carbenicillin overnight at 37°C. The cultures were combined in T7 promotor and coding region of PsSOR and PsI3S1 in pET- a 2,000 ml baffle flask. Thereafter, 1,000 ml of the LB medium 53-DEST were amplified by PCR, and the PCR products was added with carbenicillin and 30 mg formononetin (1) were cloned into a downstream region of PsIFR cDNA in dissolved in dimethyl sulfoxide : tween 80 : ethanol=1 : 1 : 1 pET-53-DEST. (v/v/v), and supplements optimized for P450 expression The nucleotide sequence data reported herein have been (Uchida et al. 2015). Co-cultured E. coli cells were grown deposited in the DDBJ, GenBank, and EMBL databases under at 25°C for 72 h. E. coli cells were removed by centrifugation the following accession numbers: PsI3S1, LC497416; GmI3S1, (9,000 g for 1 min at 4°C), and the supernatant was extracted LC497417; LjI3S2, LC497419; LjI3S1, LC497418; and GmI4R, three times with ethyl acetate. The ethyl acetate extracts of the LC497420. reaction mixtures were analyzed by HPLC with the conditions defined as ‘program 3’ (Supplementary Data S1). For NMR Heterologous expression and in vitro enzyme analysis, the extract was concentrated in vacuo and applied assay to silica-gel TLC with the solvent toluene : ethyl acetate : The E. coli strain BL21-CodonPlus (DE3)-RIPL (Agilent methanol : light petroleum (6 : 4 : 1 : 3, v/v/v/v) and the product Technologies) harboring an expression vector integrated (Rf value=0.49) was subsequently collected. with N-terminus truncated I3S (PsI3S1, LjI3S1, LjI3S2, and GmI3S1), were cultured overnight in 10 ml Luria Broth (LB) Results liquid medium containing 50 mg l−1 carbenicillin and 30 mg l−1 chloramphenicol. The cells were collected by centrifugation RNA-sequence analysis and selection of the I3S (9,000 g for 1 min at 4°C) and re-suspended in 200 ml terrific candidate gene broth liquid medium containing 1% glucose, 2% ethanol, RNA-sequence and de novo assemble generated 47,799 50 mg l−1 carbenicillin, and 0.1 mM IPTG. After incubation for contigs. When TBLASTN was employed using GePTS1 24 h at 18°C, E. coli cells were harvested, and the recombinant as the query, 5 contigs that share >50% identity were I3S proteins were purified according to a previous report obtained (Supplementary Figure S1). Among them, (Akashi et al. 2003). The enzyme assay was carried out in 0.1 M Pisum21_contig00001700 had the highest reads per potassium phosphate buffer (pH 6.5) at 30°C in a total volume kilobase of exon model per million mapped reads of 300 µl. For the initial assay, the purified PsI3S1 protein (ca. (RPKM) value and seemed to contain the full-length 2 µg) and the crude protein extract of E. coli cells (ca. 200 µg) coding sequence. The corresponding cDNA was cloned expressing GmI3S1, LjI3S1, and LjI3S2 were incubated for by RT-PCR and tentatively referred to as PsI3S1 for the 5 min and 30 min, respectively. To determine the relative high structural similarity (56% amino acid identity) to activity, 50 µM (3R,4R)-DMI (4a) was incubated with purified GePTS1. I3S proteins (PsI3S1, 80 ng; GmI3S, 15 ng; LjI3S1 and LjI3S2,

Biochemical analysis of PsI3S1 PsI3S1 was predicted to have the coding sequence of 657 bp and encode 218 amino acids. Most of the DIR domain-containing proteins have been reported to contain a putative signal peptide sequence at the N-terminus and processed into mature active forms after truncation of the signal peptide (Kim et al. 2002). The prediction of subcellular localization using WoLFPSORT (http://wolfpsort.org/) indicated that PsI3S1 is extracellular and/or vacuolar localized as well as GePTS1 (Uchida et al. 2017). Therefore, for biochemical analysis of PsI3S1, recombinant proteins were expressed as signal peptide-truncated and histidine-tagged forms in E. coli cells. Enzymatic properties were examined after affinity purification, and the assay was carried out with (3R,4R)-DMDI (4b) and (3R,4R)-DMI (4a) as substrates. The substrates are unstable and rapidly convert to pterocarpans under acidified conditions; therefore, 0.1 M potassium phosphate buffer (pH 6.5), which is the same buffer as in the PTS reaction, was used for the enzyme assay (Uchida et al. 2017). The incubation mixture of PsI3S1 with (3R,4R)-DMDI (4b) or (3R,4R)-DMI (4a) gave a single product on HPLC, and the products were identified as DMDIF (5b) and 7,2′-dihydroxy- Figure 2. HPLC elution profiles of the products of recombinant I3S reactions. Affinity purified PsI3S1 and crude extracts of E. coli 4′-methoxyisoflav-3-ene (DMIF, 5a), respectively, by expressing I3S proteins of soybean (GmI3S1) and L. japonicus (LjI3S1 1 comparing UV, mass, or H-NMR spectra (Figure 2, and LjI3S2) were reacted with (3R,4R)-DMI (4a), and ethyl acetate Supplementary Figure S2, Data S3). These results show extracts of the reaction mixtures were analyzed. Because the maximum that PsI3S1 possesses isoflav-3-ene synthetic activity in absorbances of the substrate (λmax 280 nm, retention time 5.6 min) and product (5a, λmax 340 nm, retention time 13.8 min) are largely different, vitro. the substrate was not detected in this chromatogram. The ordinate scales of the HPLC charts are equal. The eluates (retention times 5.0 Distribution of I3S in leguminous plants and to 6.0 min) monitored at 280 nm are shown in the dotted line frame, phylogenetic analysis and the ordinate scales represent mAU. UV spectra of the product (5a) by PsI3S1 reaction and the substrate (4a) are shown in the line frame. To examine the distribution of the I3S protein in A crude extract of E. coli transformed with pET-21a was used as the leguminous plants, I3S-like cDNAs were cloned from control. soybean and L. japonicus by RT-PCR. N-terminus truncated proteins were expressed in E. coli, and catalytic confirmed PTS and I3S showed that the I3S proteins have activity was tested using crude protein extracts with a C-terminus region that is 9–25 aa longer than that of (3R,4R)-DMI (4a) as the substrate. As a result, the I3S PTS proteins (Figure 3A). As the postulated substrate activity of three orthologues, GmPTS-L4 from soybean and hypothetical reaction mechanism of I3S were the and LjPTS-L1 and LjPTS-L2 from L. japonicus, were same as or very similar to those of PTS, a phylogenetic confirmed (designated as GmI3S1, LjI3S2, and LjI3S1, tree was constructed using the amino acid sequence of respectively) (Figure 2). The specific activity of I3S DIR domain-containing proteins, in particular the DIR- proteins toward (3R,4R)-DMI (4a) was compared using b/d subfamily (Uchida et al. 2017). Phylogenetic analysis the purified recombinant proteins. GmI3S1 showed the showed that the I3S proteins constituted a distinct clade highest specific activity (151.6±0.8 µmol min−1 mg−1; from that of the PTS (Figure 3B). relative activity, 100%). PsI3S1, LjI3S1, and LjI3S2 showed 5%, 46%, and 53% of the activity of GmI3S1, Time courses of (+)-pisatin-accumulation and respectively. The pH dependence of I3S activity was transcript levels of the biosynthetic genes in also examined using GmI3S1 and (3R,4R)-DMI. elicited pea cotyledon The optimum pH range was pH 6.0–8.0, and activity To verify whether PsI3S1 participates in (+)-pisatin decreased rapidly outside this range (Supplementary biosynthesis, the accumulation of (+)-pisatin (7c) and Figure S3). the expression of its biosynthetic genes were analyzed.

The identity of amino acid sequences among I3S An abiotic stress agent, CuCl2, activates isoflavonoid proteins was 55–87% (Supplementary Data S4). metabolism in leguminous plants (Dewick 1986).

Alignment of the amino acid sequence of the function- (+)-Pisatin (7c) production was induced by 5 mM CuCl2

Figure 3. Alignment of amino acid sequences and the phylogenetic relationships among I3S, PTS, and the related DIR domain-containing proteins. (A) The amino acid residues with at least four identical sequences are in the reverse type. Gaps (−) are inserted to optimize alignment. (B) Evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees where the associated taxa are clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Accession numbers of proteins are listed in Supplementary Data S6.

Figure 4. Time course of (+)-pisatin accumulation (A) and transcript levels of the biosynthetic genes (B) in pea cotyledon upon CuCl2 treatment. Data are expressed as mean±SE (n=3 biological replicates). Transcript levels were analyzed using the ΔΔCt method. β-Tubulin was used as the internal standard. Transcript levels were normalized to those of non-treated cotyledons (at 0 h).

treatment, and its maximum content was observed One-pot synthesis of isoflav-3-ene from isoflavone at 24 h and 48 h after elicitation (Figure 4A). Although using a co-cultured recombinant E. coli system (+)-pisatin (7c) was also induced by the mock treatment, and biosynthesis of coumestrol in elicited its amount was more than 10-fold lower than that of the soybean cells

CuCl2-treated cotyledon. The transcript levels of PsI3S1 Isoflav-3-enes are considered to be precursors in some and (+)-pisatin biosynthetic genes (PsIFS and PsIFR) of the isoflavonoid pathways such as (+)-pterocarpan, were markedly increased by the elicitation, and the coumestan, and 2-arylbenzofuran. An effective levels were 4.8–7.5-fold higher than the mock-treated method for isoflav-3-ene production is thus important cotyledon (Figure 4B). to elucidate the biosynthetic mechanism. For the biotechnological production of isoflav-3-ene, recombinant E. coli cells co-expressing isoflavone 2′-hydroxylase (I2′H), cytochrome P450 reductase

Figure 5. One-pot synthesis of isoflav-3-ene from isoflavone. Multiple expression vector and biosynthetic scheme (A) and HPLC chromatogram (B) are shown. Upper chromatogram and lower chromatogram show 0 h and 72 h after incubation with 1a, respectively (1a: formononetin, 5a: DMIF). T7 Pro, T7 promoter; T7 Ter, T7 terminator; AmpR, ampicillin resistance gene.

(CPR), IFR, I4R, and I3S were tested for their in vivo crude extract of soybean, but two new peaks appeared in metabolism (bioconversion) from exogenously supplied the crude extract of soybean (Supplementary Figure S5). isoflavone. First, we constructed a multiple expression Based on the retention times and UV spectra, one of the vector by introducing coding sequences of PsIFR, peaks was identified to be coumestrol (8). PsSOR, and PsI3S1 of pea (Figure 5A). Recombinant E. coli cells harboring LjCPR1 and codon-optimized Discussion LjI2′H (Uchida et al. 2015) of L. japonicus or harboring PsIFR, PsSOR, and PsI3S1 were pre-cultured separately Isoflav-3-enes have been of interest because of their in LB liquid medium, and then both cultures were characteristic biological activities, and considerably combined in one flask and fermented for 72 h in the attracted attention as a key intermediate in (+)-pisatin presence of the substrate formononetin (1). As shown in biosynthesis of pea. In the present study, we identified Figure 5B, a new peak was observed by HPLC, but no I3S cDNAs from pea (PsI3S1), soybean (GmI3S1), and L. other biosynthetic intermediates were detected in this japonicus (LjI3S1 and LjI3S2). It was predicted that all I3S condition. The chemical structure of the product was proteins contained a putative signal peptide sequence at confirmed by 1H-NMR to be DMIF (5a). The amount of the N-terminus and were localized to extracellular region DMIF (5a) calculated from the peak area on HPLC was or vacuole. Although their subcellular localizations were ca. 15 mg l−1 after 72 h of fermentation. The stability of not experimentally verified, the signal peptide-truncated DMIF (5a) in the neutral buffer (pH 6.5 at 30°C) was also proteins were found to produce only isoflav-3-enes from examined. The compound was decreased by a first-order (3R,4R)-2′-hydroxyisoflavanols and functionally distinct reaction, and the half-life period under this condition from PTS proteins. The specific activities of the soybean was 136 h. and L. japonicus I3S proteins for (3R,4R)-DMI (4a) To examine the role of I3S proteins in soybean were roughly at the same level as that of recombinant and L. japonicus, the metabolism of isoflav-3-ene in PTS proteins, but PsI3S1 showed lower activity than the soybean was analyzed by an in vitro enzyme assay. The other I3S proteins (Uchida et al. 2017). (+)-Pisatin (7c) substrate, 7,2′,4′-trihydroxyisoflav-3-ene (THIF, 5c), has a methylenedioxy ring, and the bridge formation was prepared using co-cultured E. coli cells expressing is thought to occur at the early stage of biosynthesis I2′H (CYP81E18), IFR, I4R, and GmI3S1 of soybean because 14C-labeled methylenedioxylated isoflavone and LjCPR1 of L. japonicus, with daidzein (1c) as the (pseudobaptigenin, 1b) was efficiently incorporated precursor (Supplementary Figure S4 and Data S5). Then, into pisatin (Banks and Dewick 1982a) (Figure 1). THIF (5c) was incubated with crude extract of elicited Thus, it is assumed that the natural substrate of PsI3S1 soybean cells, and the reaction mixture was analyzed is (3R,4R)-DMDI (4b) (Celoy and VanEtten 2014), and by HPLC. As the substrate was labile, a contaminant the low activity of PsI3S1 toward (3R,4R)-DMI (4a) is emerged during the incubation of both the control and attributable to the substrate preference of the protein.

In general, the biosynthetic genes of phytoalexins are hydrogen at C-3 and the hydroxy group at C-4 of induced by biotic and abiotic stresses (e.g., attack by (3R,4R)-2′-hydroxyisoflavanol, contrasting the PTS- pathogens, herbivory, and elicitor treatment), and mediated reaction, which produces the chiral product are considerably upregulated prior to the increased (−)-pterocarpan from the same substrate (Uchida et al. accumulation of phytoalexins. Therefore, comparing 2017). A previous report showed that the crude extract of the time courses of (+)-pisatin (7c) accumulation and elicited pea tissues converts the cis-(−)-DMDI [(3R,4R)- transcript levels is helpful to estimate the relevant isomer (4b)] into isoflav-3-ene; however, trans-(−)- biosynthetic pathway. In the present study, transient DMDI (probably (3R,4S)-isomer) was not metabolized upregulation of PsI3S1, PsIFS, and PsIFR transcripts (Celoy and VanEtten 2014). Thus, the configuration at was clearly observed, compared to non-elicited, in the C-4 position of the substrate is likely to be critical pea cotyledon prior to (+)-pisatin (7c) accumulation for both the I3S and PTS reactions, and the abstraction (Figure 4). These results strongly support the possibility of a hydrogen at C-2′ hydroxy or C-3 of the quinone that PsI3S1 is involved in (+)-pisatin biosynthesis. The methide intermediate is expected to determine the external stimuli, such as mechanical wounding, induce product structure as catalytic outcome. As the amino phytoalexins accumulation. (+)-Pisatin (7c) production acid sequences of both I3S and PTS proteins and the was also induced by water (mock) treatment, even crystal structures of AtDIR6 and PsDRR206 proteins in though the amount was over 10-fold lower than that of the lignan pathway (Gasper et al. 2016; Kim et al. 2015) elicitor-treated cotyledons. This result indicates that are now available, future structural studies based on cutting cotyledons from seedlings promoted (+)-pisatin homology modeling and mutagenesis would lead to the (7c) accumulation. elucidation of key amino acid residues determining their Generally, structures of phytoalexin are lineage- distinct catalysis. specific, and (+)-pisatin (7c) is a characteristic Several important biosynthetic steps of (+)-pisatin product (namely, specialized metabolite) in pea. Most (7c) are yet to be resolved. The introduction of a leguminous plants such as soybean and L. japonicus methylenedioxy ring into the isoflavone skeleton, that is, produce (−)-pterocarpans and their derivatives by stress the conversion of calycosin (1d) into pseudobaptigenin responses, whereas the biosynthesis of (+)-pterocarpans (1b), was detected in the microsomal fraction of elicited remains unknown, and biosynthetic mechanisms of chickpea cells and demonstrated to be a cytochrome coumestan and 2-arylbenzofuran have also been a P450-dependent reaction (Clemens and Barz 1996). matter of debate (Kinoshita 1997; Martin and Dewick The reaction is consistent with different cytochrome 1980). Considering that coumestrol and its derivatives P450s, CYP719A, and CYP81Q, which specifically are widely distributed among leguminous plants, and catalyze the methylenedioxy ring formation for different soybean cells produce a prenylated coumestrol by plant-specialized metabolites isoquinoline alkaloids, elicitation (Yoneyama et al. 2016), our data suggest that and lignan (Ikezawa et al. 2007; Ono et al. 2006). the soybean and L. japonicus I3S proteins participate in An enzyme similar to chickpea cytochrome P450 coumestan biosynthesis in planta. In fact, the activity should be involved in (+)-pisatin biosynthesis in pea. of the conversion of THIF (5c) to coumestrol (8) was Furthermore, of particular interest is the introduction of found in the crude extract of elicited soybean cells, also (+)-chirality into the pterocarpan skeleton. The current supporting our conclusion (Supplementary Figure S5). model for the late steps of (+)-pisatin biosynthesis Oxidation at C-2 and ring closure reactions of isoflav- employs (+)-6a-hydroxymaackiain (7b) as a direct 3-ene are necessary for the formation of coumestan precursor of (+)-pisatin (7c). Although the biochemical skeleton, and indeed, coumestrol (8) production in basis for the conversion of achiral DMDIF (5b) to enzyme preparation was accompanied by the formation (+)-6a-hydroxymaackiain (7b) is yet to be elucidated, of an unknown compound. These results imply that other the oxygen of the C-6a hydroxy group of (+)-pisatin (7c)

enzymes, which are yet to be identified, are involved in is shown to be derived from the H2O molecule and not the temporally coordinated biosynthesis with I3S. the O2 molecule (Matthews et al. 1987). Recently, we identified PTS as the first DIR domain- Finally, many phytochemicals are of great interest containing protein that possesses enzymatic activity. because of their biological activities; however, their low PTS has been assumed to catalyze the dehydration availability in plant tissues is often the bottleneck for between the hydroxy groups at C-4 and C-2′ via a further investigation. Bioconversion using recombinant quinone methide intermediate. Accordingly, the 4R microbes is a potential solution for obtaining plant configuration of the substrate should be essential for metabolites of interest, and using a co-culture system the reaction, and configuration at C-3 could determine is an emerging approach for producing a variety of the stereochemistry of pterocarpan (Supplementary biochemicals (Hori et al. 2016; Zhang et al. 2015). In Figure S6) (Uchida et al. 2017). The I3S produces fact, isoflavones were efficiently converted into the end the achiral product, isoflav-3-ene, by losing the product isoflav-3-enes (Figure 5). DMIF 5a( ) could be

Copyright © 2020 Japanese Society for Plant Biotechnology K. Uchida et al. 309 recovered at a relatively high yield (ca. 80%) by silica- DiCenzo GL, VanEtten HD (2006) Studies on the late steps gel TLC, whereas THIF (5c) was unstable and easily of (+) pisatin biosynthesis: Evidence for (−) enantiomeric intermediates. Phytochemistry 67: 675–683 decomposed on silica-gel TLC (recovery, ca. 20%). In Fischer D, Ebenau-Jehle C, Grisebach H (1990) Purification the present study, TLC-purified THIF (5c) was used for and characterization of pterocarpan synthase from elicitor- the biosynthetic analysis of coumestrol, but we recently challenged soybean cell cultures. Phytochemistry 29: 2879–2882 found that the degradation and recovery (ca. 70%) of the Gasper R, Effenberger I, Kolesinski P, Terlecka B, Hofmann E, compound can be improved by using C18 octadecylsilyl Schaller A (2016) Dirigent protein mode of action revealed by column chromatography with aqueous methanol the crystal structure of AtDIR6. Plant Physiol 172: 2165–2175 solution as the solvent (data not shown). Preparation of Guo L, Paiva NL (1995) Molecular cloning and expression of alfalfa isoflav-3-ene by this system will therefore accelerate the (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis. Arch Biochem Biophys 320: 353–360 elucidation of the biosynthetic mechanism introducing Hori K, Okano S, Sato F (2016) Efficient microbial production of (+)-chirality in pea as well as coumestrol biosynthesis in stylopine using a Pichia pastoris expression system. Sci Rep 6: soybean. 22201 Ikezawa N, Iwasa K, Sato F (2007) Molecular cloning and Acknowledgements characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica. Dr. Toshio Aoki, a professor at Nihon University and a member of FEBS J 274: 1019–1035 the editorial board of JSPCMB, passed away on March 20, 2019, Ingham JL (1979) Phytoalexin production by flowers of garden pea after about two years of fighting against his illness. We dedicate (Pisum sativum). Z Naturforsch C 34: 296–298 this paper to the memory of Professor Aoki (1961–2019). Professor Ingham JL (1982) Phytoalexins from the Leguminosae. In: Bailey Hans D. VanEtten (University of Arizona), a pioneer of pisatin JA, Mansfield JW (eds) Phytoalexins. Wiley, New York, pp 21–80 biosynthetic studies and a former collaborator of one of the present Kaimoyo E, VanEtten HD (2008) Inactivation of pea genes by RNAi authors (T. Akashi), has also deceased in 2015. We thank the late supports the involvement of two similar O-methyltransferases in Dr. VanEtten for authentic samples and express a deep regret the biosynthesis of (+)-pisatin and of chiral intermediates with a on his passing. We are grateful for the cDNA clone (GMFL02- configuration opposite that found in (+)-pisatin. Phytochemistry 07-L14) provided by the National Bioresource Project–Lotus/ 69: 76–87 Glycine (https://www.legumebase.brc.miyazaki-u.ac.jp/). Finally, Kamsteeg M, Rutherford T, Sapi E, Hanczaruk B, Shahabi S, the technical assistance of Saori Takemoto and Takahide Misaki of Flick M, Brown D, Mor G (2003) Phenoxodiol—an isoflavone Nihon University is also gratefully acknowledged. analog—induces apoptosis in chemoresistant ovarian cancer cells. 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