Natural variation of ZmCGT1 is responsible for isoorientin accumulation in maize silk.

Maize (Zea mays L.) silk contains high levels of flavonoids and is widely used to promote human health. Isoorientin, a natural C-glycoside flavone abundant in maize silk, has attracted considerable attention due to its potential value. Although different classes of flavonoid have been well characterized in plants, the genes involved in the biosynthesis of isoorientin in maize are largely unknown. Here, we used targeted metabolic profiling of isoorientin on the silks in an association panel consisting of 294 maize inbred lines. We identified the gene ZmCGT1 by genome-wide association analysis. The ZmCGT1 protein was characterized as a 2-hydroxyflavanone C-glycosyltransferase that can C-glycosylate 2-hydroxyflavanone to form flavone-C-glycoside after dehydration. Moreover, ZmCGT1 overexpression increased isoorientin levels and RNA interference-mediated ZmCGT1 knockdown decreased accumulation of isoorientin in maize silk. Further, two nucleotide polymorphisms, A502C and A1022G, which led to amino acid changes I168L and E341G, respectively, were identified to be functional polymorphisms responsible for the natural variation in isoorientin levels. In summary, we identified the gene ZmCGT1, which plays an important role in isoorientin biosynthesis, providing insights into the genetic basis of the natural variation in isoorientin levels in maize silk. The identified favorable CG allele of ZmCGT1 may be further used for genetic improvement of nutritional quality in maize.

A Newly Synthesized Flavone from Luteolin Escapes from COMT-Catalyzed Methylation and Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages via JNK, p38 and NF-κB Signaling Pathways.

Luteolin is a common dietary flavone possessing potent anti-inflammatory activities. However, when administrated in vivo, luteolin becomes methylated by catechol-O-methyltransferases (COMT) owing to the catechol ring in the chemical structure, which largely diminishes its anti-inflammatory effect. In this study, we made a modification on luteolin, named LUA, which was generated by the chemical reaction between luteolin and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Without a catechol ring in the chemical structure, this new flavone could escape from the COMT-catalyzed methylation, thus affording the potential to exert its functions in the original form when administrated in the organism. Moreover, an LPS-stimulated RAW cell model was applied to detect the anti-inflammatory properties. LUA showed much more superior inhibitory effect on LPS-induced production of NO than diosmetin (a major methylated form of luteolin) and significantly suppressed upregulation of iNOS and COX-2 in macrophages. LUA treatment dramatically reduced LPS-stimulated reactive oxygen species (ROS) and mRNA levels of pro-inflammatory mediators such as IL-1ß, IL-6, IL-8 and IFN-ß. Furthermore, LUA significantly reduced the phosphorylation of JNK and p38 without affecting that of ERK. LUA also inhibited the activation of NF-κB through suppression of p65 phosphorylation and nuclear translocation.

Identification of biological pathway and process regulators using sparse partial least squares and triple-gene mutual interaction.

Identification of biological process- and pathway-specific regulators is essential for advancing our understanding of regulation and formation of various phenotypic and complex traits. In this study, we applied two methods, triple-gene mutual interaction (TGMI) and Sparse Partial Least Squares (SPLS), to identify the regulators of multiple metabolic pathways in Arabidopsis thaliana and Populus trichocarpa using high-throughput gene expression data. We analyzed four pathways: (1) lignin biosynthesis pathway in A. thaliana and P. trichocarpa; (2) flavanones, flavonol and anthocyannin biosynthesis in A. thaliana; (3) light reaction pathway and Calvin cycle in A. thaliana. (4) light reaction pathway alone in A. thaliana. The efficiencies of two methods were evaluated by examining the positive known regulators captured, the receiver operating characteristic (ROC) curves and the area under ROC curves (AUROC). Our results showed that TGMI is in general more efficient than SPLS in identifying true pathway regulators and ranks them to the top of candidate regulatory gene lists, but the two methods are to some degree complementary because they could identify some different pathway regulators. This study identified many regulators that potentially regulate the above pathways in plants and are valuable for genetic engineering of these pathways.

Analysis of secondary metabolites induced by yellowing process for understanding rice yellowing mechanism.

The current study applied wide-targeted metabolomics based approach using LC-ESI-MS/MS to characterize the secondary metabolic difference between yellowed and normal rice. The results indicated that the biosynthesis of secondary metabolites including flavonoids, flavonols and phenolic acids was significantly enhanced during the rice yellowing process, which appears to be highly managed by phenylpropanoid metabolism and flavonoid biosynthetic pathways. Furthermore, rice yellowing led to an increased color parameter b* value, and a number of increased secondary metabolites in the yellowed rice such as homoeriodictyol, naringenin chalcone, 4,2',4',6'-tetrahydroxychalcone contributed to the yellow color. These may have application as potential biomarkers for characterizing rice yellowing.

Nbnrp1 mediates Verticillium dahliae effector PevD1-triggered defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway in Nicotiana benthamiana.

PevD1, a fungal effector secreted by Verticillium dahliae, could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco plants. PevD1 could drastically induce the expression of Nbnrp1, which is an asparagine-rich protein (NRP) of Nicotiana benthamiana. Our previous research indicated that Nbnrp1 positively regulated PevD1-induced cell necrosis and disease resistance. In this study, we further investigated PevD1-induced immune responses in both wild-type (WT) and Nbnrp1-RNAi lines through RNA-seq, in order to reveal the underlying mechanism of Nbnrp1-modulated PevD1-induced disease resistance in N. benthamiana. Results showed that Nbnrp1-RNAi lines exhibited reduced PevD1-induced immune responses, like inhibiting H2O2 accumulation and MAPK phosphorylation. To silence Nbnrp1 inhibited the expression of PevD1-induced differential expression genes (DEGs) involved in pathways associated with sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction and phenylpropanoid biosynthesis, etc. It is worth noting that sesquiterpene phytoalexin capsidiol accumulation were obviously decreased in Nbnrp1-RNAi plants after PevD1 treatment, accompanied with the down-expression of EAS and EAH, which were two key genes related to capsidiol biosynthesis. These results suggested that Nbnrp1 mediates PevD1-induced defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway.

Biochemical Characterization of a Flavonoid O-methyltransferase from Perilla Leaves and Its Application in 7-Methoxyflavonoid Production.

Methylation is a common structural modification that can alter and improve the biological activities of natural compounds. O-Methyltransferases (OMTs) catalyze the methylation of a wide array of secondary metabolites, including flavonoids, and are potentially useful tools for the biotechnological production of valuable natural products. An OMT gene (PfOMT3) was isolated from perilla leaves as a putative flavonoid OMT (FOMT). Phylogenetic analysis and sequence comparisons showed that PfOMT3 is a class II OMT. Recombinant PfOMT3 catalyzed the methylation of flavonoid substrates, whereas no methylated product was detected in PfOMT3 reactions with phenylpropanoid substrates. Structural analyses of the methylation products revealed that PfOMT3 regiospecifically transfers a methyl group to the 7-OH of flavonoids. These results indicate that PfOMT3 is an FOMT that catalyzes the 7-O-methylation of flavonoids. PfOMT3 methylated diverse flavonoids regardless of their backbone structure. Chrysin, naringenin and apigenin were found to be the preferred substrates of PfOMT3. Recombinant PfOMT3 showed moderate OMT activity toward eriodictyol, luteolin and kaempferol. To assess the biotechnological potential of PfOMT3, the biotransformation of flavonoids was performed using PfOMT3-transformed Escherichia coli. Naringenin and kaempferol were successfully bioconverted to the 7-methylated products sakuranetin and rhamnocitrin, respectively, by E. coli harboring PfOMT3.

De Novo Biosynthesis of Multiple Pinocembrin Derivatives in Saccharomyces cerevisiae.

Pinocembrin derived flavones are the major bioactive compounds presented in the Lamiaceae plants that have long been of interest due to their great pharmaceutical and economical significance. Modifications on the central skeleton of the flavone moiety have a huge impact on their biological activities. However, the enzymes responsible for structure modification of most flavones are either inefficient or remain unidentified. By integrating omics analysis of Scutellaria barbata and synthetic biology tools in yeast chassis, we characterized a novel gene encoding flavone 7-O-methyltransferase (F7OMT) and discovered a new flavone 8-hydroxylase (F8H) with increased activity. We also identified a series of flavone 6-hydroxylases (F6Hs) and flavone 8-O-methyltransferases (F8OMTs) in this study. Subsequently, we constructed the biosynthetic pathway for chrysin production by assembling catalytic elements from different species and improved the titer to 10.06 mg/L. Using the established chrysin production platform, we achieved the de novo biosynthesis of baicalein, baicalin, norwogonin, wogonin, isowogonin, and moslosooflavone in yeast. Our results indicated that the combination of omics and synthetic biology can greatly speed up the efficiency of gene mining in plants and the engineered yeasts established an alternative way for the production of pinocembrin derivatives.

Expression and functional analysis of the nobiletin biosynthesis-related gene CitOMT in citrus fruit.

Nobiletin, a polymethoxy flavone (PMF), is specific to citrus and has been reported to exhibit important health-supporting properties. Nobiletin has six methoxy groups at the 3',4',5,6,7,8-positions, which are catalyzed by O-methyltransferases (OMTs). To date, researches on OMTs in citrus fruit are still limited. In the present study, a novel OMT gene (CitOMT) was isolated from two citrus varieties Satsuma mandarin (Citrus unshiu Marc.) and Ponkan mandarin (Citrus reticulata Blanco), and its function was characterized in vitro. The results showed that the expression of CitOMT in the flavedo of Ponkan mandarin was much higher than that of Satsuma mandarin during maturation, which was consistent with the higher accumulation of nobiletin in Ponkan mandarin. In addition, functional analysis showed that the recombinant protein of CitOMT had methylation activity to transfer a methyl group to 3'-hydroxy group of flavones in vitro. Because methylation at the 3'-position of flavones is vital for the nobiletin biosynthesis, CitOMT may be a key gene responsible for nobiletin biosynthesis in citrus fruit. The results presented in this study will provide new strategies to enhance nobiletin accumulation and improve the nutritional qualities of citrus fruit.

Pathway-specific enzymes from bamboo and crop leaves biosynthesize anti-nociceptive C-glycosylated flavones.

C-glycosylated flavones (CGFs) are promising candidates as anti-nociceptive compounds. The leaves of bamboo and related crops in the grass family are a largely unexploited bioresource with a wide array of CGFs. We report here pathway-specific enzymes including C-glycosyltransferases (CGTs) and P450 hydroxylases from cereal crops and bamboo species accumulating abundant CGFs. Mining of CGTs and engineering of P450s that decorate the flavonoid skeleton allowed the production of desired CGFs (with yield of 20-40 mg/L) in an Escherichia coli cell factory. We further explored the antinociceptive activity of major CGFs in mice models and identified isoorientin as the most potent, with both neuroanalgesic and anti-inflammatory effects superior to clinical drugs such as rotundine and aspirin. Our discovery of the pain-alleviating flavonoids elicited from bamboo and crop leaves establishes this previously underutilized source, and sheds light on the pathway and pharmacological mechanisms of the compounds.

A spatially-resolved approach to visualize the distribution and biosynthesis of flavones in Scutellaria baicalensis Georgi.

Imaging the spatial distributions and dynamics of flavones in heterogeneous plant tissues is significant for our understanding of plant metabolism. Here, we proposed a spatially-resolved approach to map the locations and biosynthesis of flavones in S. baicalensis. A total of 11 flavones, 5 flavone glycosides, 6 carbohydrates, and a variety of flavone synthesis-related metabolites were imaged. Most of these flavone-related metabolites presented stronger ion intensities in root phloem. The biosynthetic network of flavones and their glycosides in S. baicalensis were visualized for the first time. Moreover, we characterized the region-specific activities of four crucial enzymes in flavone synthesis pathway, including l-phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate coenzyme A ligase, and flavone synthase. In line with the spatial characteristic of flavones, all these four enzymes exhibit higher activity in the root phloem of S. baicalensis. The combination of spatially-resolved metabolites and enzymes information greatly broadens our understanding of flavone biosynthetic network.

Biosynthesis of the anti-diabetic metabolite montbretin A: glucosylation of the central intermediate mini-MbA.

Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic α-amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA). Here, we describe the sequence of reactions from mini-MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini-MbA. The UDP-dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2-glucosylation of mini-MbA to produce myricetin 3-O-(glucosyl-6'-O-caffeoyl)-glucosyl rhamnoside. Co-expression of CcUGT3 with genes for myricetin and mini-MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.

Temporal biosynthesis of flavone constituents in flax growth stages.

Previous studies showed that chalcone synthase (chs) silencing in flax (Linum usitatisimum) induces a signal transduction cascade that leads to extensive modification of plant metabolism. Result presented in the current study, performed on field grown flax plants - (across the whole vegetation period) demonstrates that, in addition to its role in tannin and lignin biosynthesis, the chs gene also participates in the regulation of flavone biosynthesis during plant growth. Apigenin and luteolin glycosides constitute the flavones, the major group of flavonoids in flax. Alterations in their levels correlate with plant growth, peaking at the flower initiation stage. Suppression of chs gene expression causes significant changes in the ratio of flavone constituents at the early stage of flax growth. A significant correlation between flavonoid 3'-hydroxylase (F3'H) gene expression and accumulation of luteolin glycosides has been found, indicating that flavone biosynthesis during flax growth and development is regulated by temporal expression of this gene. The lack of such a correlation between the flavone synthase (FNS) gene and flavone accumulation in the course of plant growth suggests that the main route of flavone biosynthesis is mediated by eriodictyol. This is the first report indicating the ratio of flavone constituents as a potent marker of flax growth stages and temporal expression of F3'H, the key gene of their biosynthesis.

Biosynthesis of cannflavins A and B from Cannabis sativa L.

In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist numerous other specialized metabolites in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as cannflavin A and cannflavin B. These prenylated flavonoids specifically accumulate in C. sativa and are known to exhibit potent anti-inflammatory activity in various animal cell models. However, almost nothing is known about their biosynthesis. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Further evidence is presented for an O-methyltransferase (CsOMT21) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin ► chrysoeriol ► cannflavin A and cannflavin B. Taken together, the identification of these two unique enzymes represent a branch point from the general flavonoid pathway in C. sativa and offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds.

Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A.

The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia × crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3'-hydroxylase (F3'H), and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence relatedness with F3'Hs in the CYP75B subfamily of cytochromes P450 than with those with known F3'5'H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg g-1 fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.

Unique Flavanol-Anthocyanin Condensed Forms in Apache Red Purple Corn.

Anthocyanin pigments from purple corn are being explored as a potential alternative to artificial colorants and for their health-promoting properties. However, all pericarp-pigmented corn varieties examined to date primarily contain cyanidin-derived anthocyanins, which produce bluish-red or pink extracts. Here we describe the first pelargonidin-dominant pericarp-pigmented corn lines from the landrace Apache Red (AR). Anthocyanins were characterized from six AR families using high-performance liquid chromatography-mass spectrometry (HPLC-MS). From this, we identified two new flavanol-anthocyanin condensed forms in corn: catechin-(4,8)-pelargonidin 3,5-diglucoside and afzelechin-(4,8)-pelargonidin 3,5-diglucoside, which were subsequently confirmed with NMR. Additionally, several apigenin-derived C-glycosyl flavones were identified in abundance. With a diverse flavonoid profile containing an array of different anthocyanin species and flavones, Apache Red will be an important line in which to study control of the flavonoid biosynthesis pathway.

Indirect and direct routes to C-glycosylated flavones in Saccharomyces cerevisiae.

BACKGROUND: C-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health. These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. In contrast to O-glycosyltransferases, few C-glycosyltransferases (CGTs) have so far been characterized. Two different biosynthetic routes for C-glycosylated flavones have been identified in plants. Depending on the type of C-glycosyltransferase, flavones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyflavanone intermediate formed by a flavanone 2-hydroxylase (F2H). RESULTS: In this study, we reconstructed the pathways in the yeast Saccharomyces cerevisiae, to produce some relevant CGT substrates, either the flavanones naringenin and eriodictyol or the flavones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyflavanone intermediates into the 6C-glucoside flavones isovitexin and isoorientin, and the 8C-glucoside flavones vitexin and orientin. Furthermore, we established direct glycosylation of flavones using the recently identified GtUF6CGT1 from Gentiana triflora. The ratio between 6C and 8C glycosylation depended on the CGT used. The indirect route resulted in mixtures, similar to what has been reported for in vitro experiments. In this case, hydroxylation at the flavonoid 3'-position shifted the ratio towards the 8C-glucosylated orientin. The direct flavone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides. CONCLUSIONS: The current study features yeast as a promising host for production of flavone C-glycosides, and it provides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation.

Biosynthesis of 7,8-dihydroxyflavone glycosides via OcUGT1-catalyzed glycosylation and transglycosylation.

Herein, a flavonoid glycosyltransferase (GT) OcUGT1 was determined to be able to attack C-8 position of 7,8-dihydroxyflavone (7,8-DHF) via both glycosylation and transglycosylation reactions. OcUGT1-catalyzed glycosylation of 7,8-DHF resulted in the formation of two monoglycosides 7-O-ß-D-glucosyl-8-hydroxyflavone (1a), 7-hydroxy-8-O-ß-D-glucosylflavone (1b), as well as one diglycoside 7,8-di-O-ß-D-glucosylflavone (1c). Under the action of OcUGT1, inter-molecular trans-glycosylations from aryl ß-glycosides to 7,8-DHF to form monoglycosides 1a and 1b were observable.

Cloning and characterization of a novel phenylalanine ammonia-lyase gene from Inonotus baumii.

Phenylalanine ammonia-lyase (PAL) gene plays an important role in the synthesis of flavones, lignin, and other bioactive compounds in living organisms. Inonotus baumii, the only known flavone-producing filamentous fungus, is of great importance in the investigation of flavone metabolic pathways. To study the function of PAL enzyme in I. baumii flavone synthesis, a full-length cDNA of pal gene was cloned from I. baumii using DOP-PCR and RACE-PCR. The 2502-bp PAL coding region encodes an 833 amino acid protein with an approximate MW of 88.2kDa. Three introns and four exons are present in the DNA sequence of IbPAL. Amino acid sequence alignment showed that IbPAL shares 76% similarity with PALs of Inonotus fungi. The three-dimensional structure of IbPAL showed that it is composed of an MIO domain, a core domain and an inserted shielding domain. On this basis, the IbPAL was expressed and purified using the prokaryotic expression vector pSMART-V with a 6xHis-tag in Escherichia coli, and its enzymatic activity was subsequently detected. Our results will aid in understanding the enzymatic properties of PAL and further confirm the mechanism of flavone synthesis in I. baumii.

Biosynthesis of flavone C-glucosides in engineered Escherichia coli.

Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of selected flavones chrysin and luteolin. Uridine diphosphate (UDP)-glucose pool was enhanced in Escherichia coli cell cytosol by introducing heterologous UDP-glucose biosynthetic genes, i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector with individual T7 promoter, ribosome binding site, and terminator for each gene. The C-glucosylated products were analyzed by high-performance liquid chromatography-photodiode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 µM) and 11% (9 mg/L; 22 µM) conversion of chrysin to chrysin 6-C-ß-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilitator diffusion protein enhanced the production of chrysin 6-C-ß-D-glucoside by approximately 1.4-fold, thus producing 10 mg/L (12%, 24 µM) and 14 mg/L (17%, 34 µM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab-scale fermentor at 3 L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 µM) conversion of chrysin to chrysin 6-C-ß-D-glucoside at 48 h upon supplementation of 200 µM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 µM) in 50-mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble and more stable to high temperature, different pH range, and ß-glucosidase enzyme, than O-glucosylated derivative of chrysin.

Molecular Basis of Overdominance at a Flower Color Locus.

Single-gene overdominance is one of the major mechanisms proposed to explain heterosis (i.e., hybrid vigor), the phenomenon that hybrid offspring between two inbred lines or varieties show superior phenotypes to both parents. Although sporadic examples of single-gene overdominance have been reported over the decades, the molecular nature of this phenomenon remains poorly understood and it is unclear whether any generalizable principle underlies the various cases. Through bulk segregant analysis, chemical profiling, and transgenic experiments, we show that loss-of-function alleles of the FLAVONE SYNTHASE (FNS) gene cause overdominance in anthocyanin-based flower color intensity in the monkeyflower species Mimulus lewisii FNS negatively affects flower color intensity by competing with the anthocyanin biosynthetic enzymes for the same substrates, yet positively affects flower color intensity by producing flavones, the colorless copigments required for anthocyanin stabilization, leading to enhanced pigmentation in the heterozyote (FNS/fns) relative to both homozygotes (FNS/FNS and fns/fns). We suggest that this type of antagonistic pleiotropy (i.e., alleles with opposing effects on different components of the phenotypic output) might be a general principle underlying single-gene overdominance.