Uncovering nutritional metabolites and candidate genes involved in flavonoid metabolism in Houttuynia cordata through combined metabolomic and transcriptomic analyses.

The perennial herb Houttuynia cordata has long been cultivated and used as medicinal and edible plant in Asia. Nowadays, increasing attention is attracted due to its numerous health benefits. Flavonoids are the main chemical constituents exerting pharmacological activities. In the present study, we investigated both metabolome and transcriptome of two H. cordata accessions (6# and 7#) with distinct flavonoids contents. In total 397 metabolites, i.e., 220 flavonoids, 92 amino acids and derivatives, 20 vitamins, and 65 saccharides were abundant in aboveground part. Cyanidin-3-O-rutinoside and quercetin-3-O-galactoside were the most abundant flavonoids, which can be categorized into seven classes, namely anthocyanidins, chalcones, flavanols, flavanones, flavanonols, flavones, and flavonols. Flavonols was the most abundant group. Contents of 112 flavonoids differed significantly between the two accessions, with catechin-(7,8-bc)-4α-(3,4-dihydroxyphenyl)-dihydro-2-(3H)-one, cinchonain Id, and cinchonain Ic being the dominant flavonoid metabolites among them. Pinocembrin-7-O-neohesperidoside, pinocembrin-7-O-rutinoside, and kaempferol-3-O-galactoside-4'-O-glucoside were uniquely abundant in accession 7. Transcriptome data revealed a total of 110 different expressed genes related to flavonoid metabolism, with more highly expressed genes observed in 7#. We annotated a total of 19 differential flavonoid metabolites and 34 differentially expressed genes that are associated with the flavonoid metabolic network. Based on the transcriptome and qPCR data a total of 8 key candidate genes involved in flavonoid metabolism were identified. The ANS gene were found to play an important role in the synthesis of cyanidin-3-O-glucoside, while the CHI, F3'H and FLS genes were mainly responsible for controlling the levels of flavanones, flavones, and flavonols, respectively. Collectively, the present study provides important insights into the molecular mechanism underlying flavonoid metabolism in H. cordata.

Riboswitch-guided chalcone synthase engineering and metabolic flux optimization for enhanced production of flavonoids.

Flavonoids are a group of secondary metabolites from plants that have received attention as high value-added pharmacological substances. Recently, a robust and efficient bioprocess using recombinant microbes has emerged as a promising approach to supply flavonoids. In the flavonoid biosynthetic pathway, the rate of chalcone synthesis, the first committed step, is a major bottleneck. However, chalcone synthase (CHS) engineering was difficult because of high-level conservation and the absence of effective screening tools, which are limited to overexpression or homolog-based combinatorial strategies. Furthermore, it is necessary to precisely regulate the metabolic flux for the optimum availability of malonyl-CoA, a substrate of chalcone synthesis. In this study, we engineered CHS and optimized malonyl-CoA availability to establish a platform strain for naringenin production, a key molecular scaffold for various flavonoids. First, we engineered CHS through synthetic riboswitch-based high-throughput screening of rationally designed mutant libraries. Consequently, the catalytic efficiency (kcat/Km) of the optimized CHS enzyme was 62% higher than that of the wild-type enzyme. In addition to CHS engineering, we designed genetic circuits using transcriptional repressors to fine-tune the malonyl-CoA availability. The best mutant with synergistic effects of the engineered CHS and the optimized genetic circuit produced 98.71 mg/L naringenin (12.57 mg naringenin/g glycerol), which is the highest naringenin concentration and yield from glycerol in similar culture conditions reported to date, a 2.5-fold increase compared to the parental strain. Overall, this study provides an effective strategy for efficient production of flavonoids.

Identification and characterization of unique 5-hydroxyisoflavonoid biosynthetic key enzyme genes in Lupinus albus.

KEY MESSAGE: 5-Hydroxyisoflavonoids, no 5-deoxyisoflavonoids, in Lupinus species, are due to lack of CHRs and Type II CHIs, and the key enzymes of isoflavonoid biosynthetic pathway in white lupin were identified. White lupin (Lupinus albus) is used as food ingredients owing to rich protein, low starch, and rich bioactive compounds such as isoflavonoids. The isoflavonoids biosynthetic pathway in white lupin still remains unclear. In this study, only 5-hydroxyisoflavonoids, but no 5-deoxyisoflavonoids, were detected in white lupin and other Lupinus species. No 5-deoxyisoflavonoids in Lupinus species are due to lack of CHRs and Type II CHIs. We further found that the CHI gene cluster containing both Type I and Type II CHIs possibly arose after the divergence of Lupinus with other legume clade. LaCHI1 and LaCHI2 identified from white lupin metabolized naringenin chalcone to naringenin in yeast and tobacco (Nicotiana benthamiana), and were bona fide Type I CHIs. We further identified two isoflavone synthases (LaIFS1 and LaIFS2), catalyzing flavanone naringenin into isoflavone genistein and also catalyzing liquiritigenin into daidzein in yeast and tobacco. In addition, LaG6DT1 and LaG6DT2 prenylated genistein at the C-6 position into wighteone. Two glucosyltransferases LaUGT1 and LaUGT2 metabolized genistein and wighteone into its 7-O-glucosides. Taken together, our study not only revealed that exclusive 5-hydroxyisoflavonoids do exist in Lupinus species, but also identified key enzymes in the isoflavonoid biosynthetic pathway in white lupin.

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins.

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Activation of Naringenin and Kaempferol through Pathway Refactoring in the Endophyte Phomopsis Liquidambaris.

Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR, respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes.

In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes.

Transglycosylation toward naringenin-7-O-glucoside using an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase.

Flavonoids are generally glycosylated, and the glycan moieties of flavonoid glycosides are known to greatly affect their physicochemical and biological properties. Thus, the development of a variety of tools for glycan remodeling of flavonoid glycosides is highly desired. An endo-ß-N-acetylglucosaminidase mutant Endo-CC N180H, which is developed as an excellent chemoenzymatic tool for creating sialylglycoproteins, was employed for the glycosylation of flavonoids. Endo-CC N180H transferred the sialyl biantennary glycans from the sialylglyco peptide to pNP-GlcNAc and narigenin-7-O-glucoside. The kinetic parameters of Endo-CC N180H towards SGP and pNP-GlcNAc were determined. Flavonoid glucosides harboring a 1,3-diol structure in the glucose moieties acted as substrates of Endo-CC N180H. We proposed that the sialyl biantennary glycan transfer to the flavonoid by Endo-CC N180H could pave the way for the improvement of the inherent biological functions of the flavonoids and creation of novel flavonoid glycoside derivatives for future human health benefits including foods and drugs.

Simultaneously Quantitative Analysis of Naringin and Its Major Human Gut Microbial Metabolites Naringenin and 3-(4'-Hydroxyphenyl) Propanoic Acid via Stable Isotope Deuterium-Labeling Coupled with RRLC-MS/MS Method.

Widespread in citrus fruits, naringin, a natural 2,3-dihydroflavonoid, is of particular interest to scientists and has a broad range of beneficial bioactivities to health. Orally administered naringin remains in the gut tract for a relatively long time because of its low bioavailability. Under the metabolism mediated by human gut microbiota, naringin could be an active precursor for derived metabolites to play important physiological roles. However, naringin and its metabolites are hard to accurately quantify due to severe endogenic interference. In this study, an analytical rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) method coupled with stable isotope deuterium-labeling is developed and validated to simultaneously quantify naringin as well as its major human gut microbial metabolites naringenin and 3-(4'-hydroxyphenyl) propanoic acid. By eliminating the matrix interferences, this strategy not only confirms naringenin and 3-(4'-hydroxyphenyl) propanoic acid as the predominant metabolites which contribute to the pharmacological effects of naringin but also provides a suitable choice for other flavonoid pharmacokinetics study.

The Modification of the Flavonoid Naringenin by Bradyrhizobium sp. Strain ORS285 Changes the nod Genes Inducer Function to a Growth Stimulator.

As inducers of nodulation (nod) genes, flavonoids play an important role in the symbiotic interaction between rhizobia and legumes. However, in addition to the control of expression of nod genes, many other effects of flavonoids on rhizobial cells have been described. Here, we show that the flavonoid naringenin stimulates the growth of the photosynthetic Bradyrhizobium sp. strain ORS285. This growth-stimulating effect was still observed for strain ORS285 with nodD1, nodD2, or the naringenin-degrading fde operon deleted. Phenotypic microarray analysis indicates that in cells grown in the presence of naringenin, the glycerol and fatty acid metabolism is activated. Moreover, electron microscopic and enzymatic analyses show that polyhydroxy alkanoate metabolism is altered in cells grown in the presence of naringenin. Although strain ORS285 was able to degrade naringenin, a fraction was converted into an intensely yellow-colored molecule with an m/z (+) of 363.0716. Further analysis indicates that this molecule is a hydroxylated and O-methylated form of naringenin. In contrast to naringenin, this derivative did not induce nod gene expression, but it did stimulate the growth of strain ORS285. We hypothesize that the growth stimulation and metabolic changes induced by naringenin are part of a mechanism to facilitate the colonization and infection of naringenin-exuding host plants.

The Reference Genome Sequence of Scutellaria baicalensis Provides Insights into the Evolution of Wogonin Biosynthesis.

Scutellaria baicalensis Georgi is important in Chinese traditional medicine where preparations of dried roots, "Huang Qin," are used for liver and lung complaints and as complementary cancer treatments. We report a high-quality reference genome sequence for S. baicalensis where 93% of the 408.14-Mb genome has been assembled into nine pseudochromosomes with a super-N50 of 33.2 Mb. Comparison of this sequence with those of closely related species in the order Lamiales, Sesamum indicum and Salvia splendens, revealed that a specialized metabolic pathway for the synthesis of 4'-deoxyflavone bioactives evolved in the genus Scutellaria. We found that the gene encoding a specific cinnamate coenzyme A ligase likely obtained its new function following recent mutations, and that four genes encoding enzymes in the 4'-deoxyflavone pathway are present as tandem repeats in the genome of S. baicalensis. Further analyses revealed that gene duplications, segmental duplication, gene amplification, and point mutations coupled to gene neo- and subfunctionalizations were involved in the evolution of 4'-deoxyflavone synthesis in the genus Scutellaria. Our study not only provides significant insight into the evolution of specific flavone biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants. The reference genome of S. baicalensis is also useful for improving the genome assemblies for other members of the mint family and offers an important foundation for decoding the synthetic pathways of bioactive compounds in medicinal plants.

Fine-tuning the (2S)-naringenin synthetic pathway using an iterative high-throughput balancing strategy.

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry-fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl3 and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.

Inheritance of carthamin and carthamidin in safflower (Carthamus tinctorius L.).

The safflower (Carthamus tinctorius L.) is an oil seed crop from which the flowers is used as medicine and food colorants. The present investigation was undertaken to explore gene effects for safflower's pigments in flower including carthamin and carthamidin. Six generation including P1, P2, F1, F2, BC1 and BC2 that derived from two different crosses (Mex. 2-138 (P2) × Wht-Esf (P1) and C111 (P2) × Wht-Esf (P1) were used for generation of mean analysis. The joint scaling test showed that additive [a], additive × additive [aa], and additive × dominance [ad] effects were significant for genetic control of carthamin and carthamidin in both crosses. The traits, including carthamidin and carthamin, had medium (48%) and low (17%) narrow-sense heritability, respectively. The results obtained here could be suitable for designing the breeding strategies based on selection to improve carthamin and carthamidin pigments in safflower.

Full-Length Transcriptome Sequencing and Modular Organization Analysis of the Naringin/Neoeriocitrin-Related Gene Expression Pattern in Drynaria roosii.

Drynaria roosii (Nakaike) is a traditional Chinese medicinal fern, known as 'GuSuiBu'. The effective components, naringin and neoeriocitrin, share a highly similar chemical structure and medicinal function. Our HPLC-tandem mass spectrometry (MS/MS) results showed that the accumulation of naringin/neoeriocitrin depended on specific tissues or ages. However, little was known about the expression patterns of naringin/neoeriocitrin-related genes involved in their regulatory pathways. Due to a lack of basic genetic information, we applied a combination of single molecule real-time (SMRT) sequencing and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of D. roosii. According to the SGS data, the differentially expressed gene (DEG)-based heat map analysis revealed that naringin/neoeriocitrin-related gene expression exhibited obvious tissue- and time-specific transcriptomic differences. Using the systems biology method of modular organization analysis, we clustered 16,472 DEGs into 17 gene modules and studied the relationships between modules and tissue/time point samples, as well as modules and naringin/neoeriocitrin contents. We found that naringin/neoeriocitrin-related DEGs distributed in nine distinct modules, and DEGs in these modules showed significantly different patterns of transcript abundance to be linked to specific tissues or ages. Moreover, weighted gene co-expression network analysis (WGCNA) results further identified that PAL, 4CL and C4H, and C3H and HCT acted as the major hub genes involved in naringin and neoeriocitrin synthesis, respectively, and exhibited high co-expression with MYB- and basic helix-leucine-helix (bHLH)-regulated genes. In this work, modular organization and co-expression networks elucidated the tissue and time specificity of the gene expression pattern, as well as hub genes associated with naringin/neoeriocitrin synthesis in D. roosii. Simultaneously, the comprehensive transcriptome data set provided important genetic information for further research on D. roosii.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.

Deep Sequencing of the Scutellaria baicalensis Georgi Transcriptome Reveals Flavonoid Biosynthetic Profiling and Organ-Specific Gene Expression.

Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.

Modular optimization of heterologous pathways for de novo synthesis of (2S)-naringenin in Escherichia coli.

Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.

Regulation of flavanone 3-hydroxylase gene involved in the flavonoid biosynthesis pathway in response to UV-B radiation and drought stress in the desert plant, Reaumuria soongorica.

Flavonoid are known to have various functions in growth, development, reproduction, and also involved in diverse stress responses in plants. However, little is known about the roles of the key enzymes in the flavonoid biosynthetic pathway in response to environmental stress, such as UV-B radiation and drought. To understand this problem, we investigated the participation of flavanone 3-hydroxylase gene (F3H), a key enzyme in flavonoid biosynthetic pathway under UV-B radiation and drought stress in the desert plant Reaumuria soongorica. A novel cDNA sequence, named as RsF3H, was isolated from R. soongorica. The deduced amino acids showed high identities to other F3Hs. A phylogenetic analysis indicated that RsF3H appeared to be most homologous to F3H from Malus domestica (MdF3H). RsF3H protein structure contained all five conserved motifs for 2-oxoglutarate-dependent dioxygenases (2-ODDs) and an Arg-X-Ser motif, all of which were also found in other F3Hs. Quantitative real-time RT-PCR analysis showed that there was a rapid increase in gene expression of RsF3H under stress. Both UV-B radiation and drought stress induced an increase in RsF3H enzyme activity and the accumulation of the products in the flavonoid biosynthetic pathway (total flavonoid and anthocyanin). The antioxidant ability (inhibition of lipid oxidation) of total flavonoid was enhanced during this study. The results suggested that one explanation of the stress tolerance of R. soongorica may be a combination of an increase in RsF3H gene expression, RsF3H enzyme activity and the anti-oxidative ability of the metabolic end products in the flavonoid biosynthetic pathway in response to UV-B radiation and drought.

A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants.

Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy.

Flavonoids are valuable natural products widely used in human health and nutrition. Recent advances in synthetic biology and metabolic engineering have yielded improved strain titers and yields. However, current fermentation strategies often require supplementation of expensive phenylpropanoic precursors in the media and separate evaluation of each strategy in turn as part of the flavonoid pathway, implicitly assuming the modifications are additive. In this study, an Escherichia coli fermentation system was developed to bypass both of these problems. An eight-step pathway, consisting of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydratase (CM/PDT), phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was assembled on four vectors in order to produce the flavonoid precursor (2S)-pinocembrin directly from glucose. Furthermore, a modular metabolic strategy was employed to identify conditions that optimally balance the four pathway modules. Once this metabolic balance was achieved, such strains were capable of producing 40.02mg/L (2S)-pinocembrin directly from glucose. These results were attained by culturing engineered cells in minimal medium without additional precursor supplementation. The fermentation platform described here paves the way for the development of an economical process for microbial production of flavonoids directly from glucose.

Global transcriptome analysis reveals distinct expression among duplicated genes during sorghum-interaction.

BACKGROUND: Sorghum (Sorghum bicolor L. Moench) is a rich source of natural phytochemicals. We performed massive parallel sequencing of mRNA to identify differentially expressed genes after sorghum BTx623 had been infected with Bipolaris sorghicola, a necrotrophic fungus causing a sorghum disease called target leaf spot. RESULT: Seventy-six-base-pair reads from mRNAs of mock- or pathogen-infected leaves were sequenced. Unannotated transcripts were predicted on the basis of the piling-up of mapped short reads. Differentially expressed genes were identified statistically; particular genes in tandemly duplicated putative paralogs were highly upregulated. Pathogen infection activated the glyoxylate shunt in the TCA cycle; this changes the role of the TCA cycle from energy production to synthesis of cell components. The secondary metabolic pathways of phytoalexin synthesis and of sulfur-dependent detoxification were activated by upregulation of the genes encoding amino acid metabolizing enzymes located at the branch point between primary and secondary metabolism. Coordinated gene expression could guide the metabolic pathway for accumulation of the sorghum-specific phytochemicals 3-deoxyanthocyanidin and dhurrin. Key enzymes for synthesizing these sorghum-specific phytochemicals were not found in the corresponding region of the rice genome. CONCLUSION: Pathogen infection dramatically changed the expression of particular paralogs that putatively encode enzymes involved in the sorghum-specific metabolic network.