Probing the Deoxyflavonoid Biosynthesis: Naringenin Chalcone Is a Substrate for the Reductase Synthesizing Isoliquiritigenin.

Isoliquiritigenin formation is a key reaction during deoxyflavonoid biosynthesis, which is catalyzed by two enzymes, chalcone synthase (CHS) and reductase (CHR). The substrates for CHS are established. However, the substrate for CHR is unknown. In this study, an in vitro reaction was performed to confirm whether naringenin chalcone can be a substrate. Naringenin chalcone was used as a substrate during the CHR reaction. Analyzing the product revealed that isoliquiritigenin was produced from naringenin chalcone, indicating that naringenin chalcone is a substrate. This study is the first to identify a substrate for CHR, reveals that deoxyflavonoid biosynthesis diverges from naringenin chalcone, endorses the term "chalcone reductase," and answers the long-standing questions about doubly-labeled acetic acid uptake pattern in deoxyflavonoid biosynthesis.

A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2-hydroxyisoflavanone synthase.

Soybean (Glycine max) 5-deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5-Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5-deoxyisoflavonoid biosynthesis because it catalyzes the production of 6'-deoxychalcone through its effects on the chalcone synthase (CHS)-catalyzed reaction. The soybean genome encodes at least 11 CHR-related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5-deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed in vitro and in planta. In the in vitro assays, the ratio of CHR products (6'-deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein-protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2-hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro-compartmentalization of isoflavone biosynthesis through the formation of an IFS-mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5-deoxyisoflavonoids in soybean roots.

Dahlia variabilis cultivar 'Seattle' as a model plant for anthochlor biosynthesis.

We investigated the bi-colored dahlia cultivar 'Seattle', which exhibits bright yellow petals with white tips, for its potential use as a model system for studies of the anthochlor biosynthesis. The yellow base contained high amounts of the 6'-deoxychalcones and the structurally related 4-deoxyaurones, as well as flavones. In contrast, only traces of anthochlors and flavones were detected in the white tips. No anthocyanins, flavonols, flavanones or dihydroflavonols were found in the petals. Gene expression studies indicated that the absence of anthocyanins in the petals is caused by a lack of flavanone 3-hydroxylase (FHT) expression, which is accompanied by a lack of expression of the bHLH transcription factor IVS. Expression of other genes involved in anthocyanidin biosynthesis such as dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) was not affected. The yellow and white petal parts showed significant differences in the expression of chalcone synthase 2 (CHS2), which is sufficient to explain the absence of yellow pigments in the white tips. Transcriptomes of both petal parts were de novo assembled and three candidate genes for chalcone reductase (CHR) were identified. None of them showed a significantly higher expression in the yellow base compared to the white tips. In summary, it was shown that the bicolouration is most likely caused by a bottleneck in chalcone formation in the white tip. The relative prevalence of flavones compared to the anthochlors in the white tips could be an indication for the presence of a so far unknown differentially expressed CHR.

Diversion of metabolic flux towards 5-deoxy(iso)flavonoid production via enzyme self-assembly in Escherichia coli.

5-Deoxy(iso)flavonoids are structural representatives of phenylpropanoid-derived compounds and play critical roles in plant ecophysiology. Recently, 5-deoxy(iso)flavonoids gained significant interest due to their potential applications as pharmaceuticals, nutraceuticals, and food additives. Given the difficulties in their isolation from native plant sources, engineered biosynthesis of 5-deoxy(iso)flavonoids in a microbial host is a highly promising alternative approach. However, the production of 5-deoxy(iso)flavonoids is hindered by metabolic flux imbalances that result in a product profile predominated by non-reduced analogues. In this study, GmCHS7 (chalcone synthase from Glycine max) and GuCHR (chalcone reductase from Glycyrrhizza uralensis) were preliminarily utilized to improve the CHR ratio (CHR product to total CHS product). The use of this enzyme combination improved the final CHR ratio from 39.7% to 50.3%. For further optimization, a protein-protein interaction strategy was employed, basing on the spatial adhesion of GmCHS7:PDZ and GuCHR:PDZlig. This strategy further increased the ratio towards the CHR-derived product (54.7%), suggesting partial success of redirecting metabolic flux towards the reduced branch. To further increase the total carbon metabolic flux, 15 protein scaffolds were programmed with stoichiometric arrangement of the three sequential catalysts GmCHS7, GuCHR and MsCHI (chalcone isomerase from Medicago sativa), resulting in a 1.4-fold increase in total flavanone production, from 69.4 mg/L to 97.0 mg/L in shake flasks. The protein self-assembly strategy also improved the production and direction of the lineage-specific compounds 7,4'-dihydroxyflavone and daidzein in Escherichia coli. This study presents a significant advancement of 5-deoxy(iso)flavonoid production and provides the foundation for production of value-added 5-deoxy(iso)flavonoids in microbial hosts.

De novo biosynthesis of liquiritin in Saccharomyces cerevisiae.

Liquiritigenin (LG), isoliquiritigenin (Iso-LG), together with their respective glycoside derivatives liquiritin (LN) and isoliquiritin (Iso-LN), are the main active flavonoids of Glycyrrhiza uralensis, which is arguably the most widely used medicinal plant with enormous demand on the market, including Chinese medicine prescriptions, preparations, health care products and even food. Pharmacological studies have shown that these ingredients have broad medicinal value, including anti-cancer and anti-inflammatory effects. Although the biosynthetic pathway of glycyrrhizin, a triterpenoid component from G. uralensis, has been fully analyzed, little attention has been paid to the biosynthesis of the flavonoids of this plant. To obtain the enzyme-coding genes responsible for the biosynthesis of LN, analysis and screening were carried out by combining genome and comparative transcriptome database searches of G. uralensis and homologous genes of known flavonoid biosynthesis pathways. The catalytic functions of candidate genes were determined by in vitro or in vivo characterization. This work characterized the complete biosynthetic pathway of LN and achieved the de novo biosynthesis of liquiritin in Saccharomyces cerevisiae using endogenous yeast metabolites as precursors and cofactors for the first time, which provides a possibility for the economical and sustainable production and application of G. uralensis flavonoids through synthetic biology.

Genome-Wide Identification of Chalcone Reductase Gene Family in Soybean: Insight into Root-Specific GmCHRs and Phytophthora sojae Resistance.

Soybean (Glycine max [L.] Merr) is one of the main grain legumes worldwide. Soybean farmers lose billions of dollars' worth of yield annually due to root and stem rot disease caused by the oomycete Phytophthora sojae. Many strategies have been developed to combat the disease, however, these methods have proven ineffective in the long term. A more cost effective and durable approach is to select a trait naturally found in soybean that can increase resistance. One such trait is the increased production of phytoalexin glyceollins in soybean. Glyceollins are isoflavonoids, synthesized via the legume-specific branch of general phenylpropanoid pathway. The first key enzyme exclusively involved in glyceollin synthesis is chalcone reductase (CHR) which coacts with chalcone synthase for the production of isoliquiritigenin, the precursor for glyceollin biosynthesis. Here we report the identification of 14 putative CHR genes in soybean where 11 of them are predicted to be functional. Our results show that GmCHRs display tissue-specific gene expression, and that only root-specific GmCHRs are induced upon P. sojae infection. Among 4 root-specific GmCHRs, GmCHR2A is located near a QTL that is linked to P. sojae resistance suggesting GmCHR2A as a novel locus for partial resistance that can be utilized for resistance breeding.

A cis-element responsible for cGMP in the promoter of the soybean chalcone synthase gene.

The cyclic nucleotides cGMP and cAMP have been reported to play key roles in the regulation of plant processes and responses. We have previously reported that several genes encoding flavonoid biosynthetic enzymes, including chalcone synthase (CHS) in soybean (Glycine max L.), were induced by cGMP but not cAMP. The soybean genome contains nine CHS gene copies (GmCHS1-9). We investigated the responsiveness of several GmCHS genes to cGMP, cAMP, NO, and white light. Quantitative RT-PCR analysis showed that the transcript levels of GmCHS7 and GmCHS8 were increased by 3.6- and 3.8-fold, respectively, with cGMP whereas the transcript levels of GmCHS2 remained constant. Although cAMP had no effect on the transcript levels of the three genes, NO had an activation effect on all three. White light activated the three genes in a transient manner, with GmCHS2, GmCHS7, and GmCHS8 transcript levels increasing 3-fold after 3 h and decreasing to basal levels after 9 h. The GmCHS8 promoter contains several important cis-elements, including the G-box and H-box forming the Unit-I-like sequence and the MYB binding sequence, a target of the GmMYB176 transcription factor regulating the expression of GmCHS8. A transient gene expression assay revealed the activation of the Unit-I-like sequence, but not of the MYB binding sequence, by cGMP. The combination of G-box and H-box was necessary for cGMP responsiveness. Taken together, these results suggest that the Unit-I-like sequence in the promoters of GmCHS7 and GmCHS8 is a cGMP responsive cis-element in these genes and that NO exerts its effect via cis-elements other than the Unit-I-like sequence.