Characterization of phenylalanine ammonia lyase and revealing flavonoid biosynthesis in Gymnema sylvestre R. Br through transcriptomic approach.

BACKGROUND: Gymnema sylvestre R.Br. is famous medicinal plant among diabetics for its gymnemic acid content. It also contains flavonoids, which are an essential component in various other products. Though some molecular information on the biosynthesis of gymnemic acid, polyoxypregnane, micro RNAs and photosynthetic efficiency is available, there is no gene level information available on the biosynthesis of flavonoids in this plant. RNA was extracted from winter-collected Gymnema sylvestre leaves and cDNA libraries were prepared and used for next generation sequencing. De novo transcriptome assembly were prepared and Coding DNA Sequences (CDS) of 13 major genes involved in flavonoids biosynthesis were identified from transcriptome data. Phenylalanine ammonia lyase gene containing full-length CDS was employed for in silico protein modelling and subsequent quality assessment. These models were then compared against publicly available databases. To confirm the identification of these genes, a similarity search was conducted using the NCBI BLAST tool. RESULTS: Therefore, in the present study, an effort has been made to provide molecular insights into flavonoid biosynthesis pathway by examining the expressed transcripts in G.sylvestre. Gene sequences of total thirteen major genes viz., phenylalanine ammonia lyase, 4-coumarate CoA ligase, cinnamic acid 4-hydroxylase, shikimate O-hydroxycinnamoyl transferase, coumaroyl quinate (coumaroyl shikimate) 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, naringenin 3-dioxygenase, flavanol synthase, flavonoid 3'-monooxygenase, Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase and leucoanthocyanidin dioxygenase were identified and a putative pathway of flavonoids biosynthesis has been illustrated based on transcriptome data. CONCLUSIONS: This transcriptome study has contributed gene-level insights into the biosynthesis of flavonoids in plants as a whole and represents the first report within a non-model plant, Gymnema sylvestre perticullarly.

Biochemical Pathways of Salicylic Acid Derived from l-Phenylalanine in Plants with Different Basal SA Levels.

As a plant hormone, salicylic acid (SA) has diverse regulatory roles in plant growth and stress resistance. Although SA is widely found in plants, there is substantial variation in basal SA among species. Tea plant is an economically important crop containing high contents of SA whose synthesis pathway remains unidentified. The phenylalanine ammonia-lyase (PAL) pathway is responsible for basal SA synthesis in plants. In this study, isotopic tracing and enzymatic assay experiments were used to verify the SA synthesis pathway in tea plants and evaluate the variation in phenylalanine-derived SA formation among 11 plant species with different levels of SA. The results indicated that SA could be synthesized via PAL in tea plants and conversion efficiency from benzoic acid to SA might account for variation in basal SA among plant species. This research lays the foundation for an improved understanding of the molecular regulatory mechanism for SA biosynthesis.

Comparative analyses reveal a phenylalanine ammonia lyase dependent and salicylic acid mediated host resistance in Zingiber zerumbet against the necrotrophic soft rot pathogen Pythium myriotylum.

Little is known about the molecular basis of host defense in resistant wild species Zingiber zerumbet (L.) Smith against the soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which causes the devastating soft rot disease in the spice crop ginger (Zingiber officinale Roscoe). We investigated the pattern of host defense between Z. zerumbet and ginger in response to P. myriotylum inoculation. Analysis of gene expression microarray data revealed enrichment of phenylpropanoid biosynthetic genes, particularly lignin biosynthesis genes, in pathogen-inoculated Z. zerumbet compared to ginger. RT-qPCR analysis showed the robust activation of phenylpropanoid biosynthesis genes in Z. zerumbet, including the core genes PAL, C4H, 4CL, and the monolignol biosynthesis and polymerization genes such as CCR, CAD, C3H, CCoAOMT, F5H, COMT, and LAC. Additionally, Z. zerumbet exhibited the accumulation of the phenolic acids including p-coumaric acid, sinapic acid, and ferulic acid that are characteristic of the cell walls of commelinoid monocots like Zingiberaceae and are involved in cell wall strengthening by cross linking with lignin. Z. zerumbet also had higher total lignin and total phenolics content compared to pathogen-inoculated ginger. Phloroglucinol staining revealed the enhanced fortification of cell walls in Z. zerumbet, specifically in xylem vessels and surrounding cells. The trypan blue staining indicated inhibition of pathogen growth in Z. zerumbet at the first leaf whorl, while ginger showed complete colonization of the pith within 36 h post inoculation (hpi). Accumulation of salicylic acid (SA) and induction of SA regulator NPR1 and the signaling marker PR1 were observed in Z. zerumbet. Silencing of PAL in Z. zerumbet through VIGS suppressed downstream genes, leading to reduced phenylpropanoid accumulation and SA level, resulting in the susceptibility of plants to P. myriotylum. These findings highlight the essential role of PAL-dependent mechanisms in resistance against P. myriotylum in Z. zerumbet. Moreover, our results suggest an unconventional role for SA in mediating host resistance against a necrotroph. Targeting the phenylpropanoid pathway could be a promising strategy for the effective management of P. myriotylum in ginger.

Interactive effects of selenium and arsenic on phenolic constituents and antioxidant activity in rice (Oryza sativa L.).

Arsenic (As) is a heavy toxic metalloid found in air, water and soil that adversely affects the plant growth by inducing oxidative stress in plants. Its contamination of rice is a serious problem throughout the world. Selenium (Se) is a beneficial micronutrient for plants that acts as an antioxidant at low doses and protect the plants against number of environmental stresses either by modulating the primary metabolic pathways or regulating the production of phenolic compounds. In the present investigation, effect of Se on different phenolics, enzymes related to their metabolism and antioxidative potential were studied in As stressed rice leaves. Rice plants were grown in pots containing sodium arsenate (2-10 mg As(V) kg-1 soil) and sodium selenate (0.5-1 mg Se kg-1 soil), both alone and in combination and leaf samples were analyzed for various biochemical parameters. Phenolic constituents increased in rice leaves with As(V) treatment from 2 to 5 mg kg-1 soil and leaves exposed to As(V) @ 5 mg kg-1 soil exhibited 1.7, 1.9 and 2.5 fold increase in total phenolics, o-dihydroxyphenols and flavonols, respectively at grain filling stage. Binary application of Se + As improved various phenolic constituents, FRAP, reducing power and antioxidant activities as compared to control. PAL, TAL and PPO activities increased from 1.3 to 4.6 fold in combined As + Se treatment at both the stages. Anthocyanin contents showed a decline (10.8 fold) with increasing As doses and its content improved at both the stages with maximum increase of 3.76 fold with As5+Se1 combination. Binary application of As + Se improved gallic acid, chlorogenic acid, 3-hydroxy benzoic acid and kaempferol contents than control whereas catechin and coumaric acid showed the reverse trend. Application of Se can modulate phenolic constituents in leaf and grains of rice Cv PR126 due to As stress that helped plants to adapt to excess As and resulted in improved plant growth.

Nutrition management of PKU with pegvaliase therapy: update of the web-based PKU nutrition management guideline recommendations.

BACKGROUND: The web-based GMDI/SERN PKU Nutrition Management Guideline, published before approval of pegvaliase pharmacotherapy, offers guidance for nutrition management of individuals with phenylketonuria (PKU) treated with dietary therapy and/or sapropterin. An update of this guideline aims to provide recommendations that improve clinical outcomes and promote consistency and best practice in the nutrition management of individuals with PKU receiving pegvaliase therapy. Methodology includes: formulation of a research question; review, critical appraisal, and abstraction of peer-reviewed studies and unpublished practice literature; expert input through Delphi surveys and a Nominal Group process; and external review by metabolic experts. RESULTS: Recommendations, summary statements, and strength of evidence are included for each of the following topics: (1) initiating a pegvaliase response trial, (2) monitoring therapy response and nutritional status, (3) managing pegvaliase treatment after response to therapy, (4) education and support for optimal nutrition with pegvaliase therapy, and (5) pegvaliase therapy during pregnancy, lactation, and adolescence. Findings, supported by evidence and consensus, provide guidance for nutrition management of individuals receiving pegvaliase therapy for PKU. Recommendations focus on nutrition management by clinicians, as well as the challenges for individuals with PKU as a result of therapy changes. CONCLUSIONS: Successful pegvaliase therapy allows the possibility for individuals with PKU to consume an unrestricted diet while still maintaining the benefits of blood phenylalanine control. This necessitates a perspective change in education and support provided to individuals in order to achieve healthy nutrient intake that supports optimal nutritional status. The updated guideline, and companion Toolkit for practical implementation of recommendations, is web-based, allowing for utilization by health care providers, researchers, and collaborators who advocate and care for individuals with PKU. These guidelines are meant to be followed always taking into account the provider's clinical judgement and considering the individual's specific circumstances. Open access is available at the Genetic Metabolic Dietitians International ( https://GMDI.org ) and Southeast Regional Genetics Network ( https://managementguidelines.net ) websites.

Detection of gamma radiation processed onion during storage using propidium iodide based fluorescence microscopy.

The determination of absorbed dose in gamma radiation processed onion (treated with 20-100 Gy for sprout inhibition) during storage is an important regulatory requirement to control unfair practices. To address this problem, a microscopy based method was developed using propidium iodide (PI) staining of onion adaxial epidermis. A proportional radiation dose dependent increase in nuclei count was observed during ambient (26 ± 2 °C) and low (2 ± 1 °C) temperature storage. The method was validated and dose of radiation could be determined accurately in stored onions using blind tests. During mechanism studies, boron-dipyrromethene (BODIPY) dye staining and malondialdehyde (MDA) estimation showed dose dependent increase in peroxidation of membrane lipids. The fluorescein diacetate (FDA) stained onion adaxial epidermis showed decrease in fluorescence indicating lowering of physiological activity. Enzyme peroxidase (POD), phenylalanine ammonia lyase (PAL) activities and phytochemicals (phenolics, flavonoids, ascorbic acid and pyruvic acid) did not change significantly with increasing dose of gamma radiation.

Biochemical defense mechanism associated with host-specific disease resistance pathways against Rhizoctonia solani AG3-PT potatoes canker disease.

MAIN CONCLUSION: Screening for resistance in 40 potato genotypes to Rhizoctonia solani AG-3PT-stem-canker, antioxidant enzymes activity as well as total phenol compounds were documented. Rhizoctonia solani AG-3PT-stem-canker is one of the most devastating diseases that leads to severe economic losses in potatoes, Solanum tuberosum globally. Crop management and eugenic practices, especially the use of resistance can be effective in reducing the disease incidence. However, the information about potato-R. Solani interaction is still limited. This study explored screening for resistance in forty potato genotypes to R. solani, analyzing biomass growth parameters (BGPs), as well as antioxidant enzymes activity of which peroxidase/peroxide-reductases (POXs), superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), ß-1,3-glucanase (GLU) and total phenol compounds (TPCs) were taken into account. In addition, we analyzed up-regulation of two gene markers (PR-1 and Osmotin), using reverse transcription quantitative PCR (RT-qPCR). For which, the resistant 'Savalan', partially resistant 'Agria', partially susceptible 'Sagita' and susceptible 'Pashandi' were selected to explore the trails in their roots and leaves over the time courses of 1, 2 and 3-weeks post inoculation (wpi) following inoculation. Cluster analysis divided potatoes into four distinct groups, based on disease severity scales (0-100%) significance. The BGPs, shoot and root length, fresh and dry weight, and root volume were also significantly higher in infected potatoes compared to non-inoculated controls. Antioxidant enzymes activity also indicated the highest increased levels for POX (fourfold at 3wpi), CAT (1.5-fold at 3wpi), SOD (6.8-fold at 1wpi), and PAL (2.7-fold at 3wpi) in the resistant genotype, 'Savalan', whereas the highest activity was recorded in TPC (twofold at 1 wpi), PPO (threefold at 3wpi), and GLU (2.3-fold at 1wpi) in partially resistant genotypes. Although the defense-related enzymatic activities were sharply elevated in the resistant and partially resistant genotypes following inoculation, no significant correlations were between the activity trends of the related enzymes. The two related gene markers also showed comprehensive transcriptional responses up to 3.4-fold, predominantly in resistant genotypes. Surprisingly, the PR-1 gene marker, basically resistant to Wilting agent Verticillium dahlia was overexpressed in resistant 'Savalan' and 'Agria' against R. solani AG3-PT. Similar results were obtained on Osmotin gene marker resistant to late-blight P. infestans, and early-blight Alternaria solani that similarly modulates immunity against R. solani. Furthermore, there was a significant correlation between resistance, enzyme activity, and gene expression in the aforesaid cultivars. Studying the physiological metabolic pathways of antioxidant enzymes activity appears to be an important direction in research to elucidate resistance to R. solani in potatoes.

Impact of sewage water irrigation on Datura innoxia grown in sandy loam soil.

BACKGROUND: A potential solution for recycling and reusing the massively produced sewage water (SW) is to irrigate certain plants instead of highly cost recycling treatment. Although the extensive and irrational application of SW may cause environmental pollution thus, continual monitoring of the redox status of the receiver plant and the feedback on its growth under application becomes an emergent instance. The impact of SW, along with well water (WW) irrigation of medicinal plant, Datura innoxia, was monitored by some physio-biochemical indices. RESULTS: The SW application amplified the growth, yield, minerals uptake, and quality of D. innoxia plants compared to the WW irrigated plants. The total chlorophyll, carotenoid, non-enzymatic antioxidants, viz. anthocyanin, flavonoids, phenolic compounds, and total alkaloids increased by 85, 38, 81, 50, 19, and 37%, respectively, above WW irrigated plants. The experiment terminated in enhanced leaf content of N, P, and K by 43, 118, and 48%, respectively. Moreover, stimulation of carbon and nitrogen metabolites in terms of proteins, soluble sugars, nitrate reductase (NR) activity, and nitric oxide (NO) content showed significant earliness in flowering time. The SW application improved not only Datura plants' quality but also soil quality. After four weeks of irrigation, the WW irrigated plants encountered nutrient deficiency-induced stress evidenced by the high level of proline, H2O2, and MDA as well as high enzyme capabilities. Application of SW for irrigation of D. innoxia plant showed the improvement of secondary metabolites regulating enzyme phenylalanine ammonia-lyase (PAL), restored proline content, and cell redox status reflecting high optimal condition for efficient cellular metabolism and performance along the experiment duration. CONCLUSIONS: These evidences approved the benefits of practicing SW to improve the yield and quality of D. innoxia and the feasibility of generalization on multipurpose plants grown in poor soil.

Characterization of phenylalanine ammonia-lyase genes facilitating flavonoid biosynthesis from two species of medicinal plant Anoectochilus.

Background: Anoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites. Methods: The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR). Results: AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis. Discussion: The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis.

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Genome-Wide Analysis and Expression Profiling of the Phenylalanine Ammonia-Lyase Gene Family in Solanum tuberosum.

Phenylalanine ammonia-lyase is one of the most widely studied enzymes in the plant kingdom. It is a crucial pathway from primary metabolism to significant secondary phenylpropanoid metabolism in plants, and plays an essential role in plant growth, development, and stress defense. Although PAL has been studied in many actual plants, only one report has been reported on potato, one of the five primary staple foods in the world. In this study, 14 StPAL genes were identified in potato for the first time using a genome-wide bioinformatics analysis, and the expression patterns of these genes were further investigated using qRT-PCR. The results showed that the expressions of StPAL1, StPAL6, StPAL8, StPAL12, and StPAL13 were significantly up-regulated under drought and high temperature stress, indicating that they may be involved in the stress defense of potato against high temperature and drought. The expressions of StPAL1, StPAL2, and StPAL6 were significantly up-regulated after MeJa hormone treatment, indicating that these genes are involved in potato chemical defense mechanisms. These three stresses significantly inhibited the expression of StPAL7, StPAL10, and StPAL11, again proving that PAL is a multifunctional gene family, which may give plants resistance to multiple and different stresses. In the future, people may improve critical agronomic traits of crops by introducing other PAL genes. This study aims to deepen the understanding of the versatility of the PAL gene family and provide a valuable reference for further genetic improvement of the potato.

A phenylalanine ammonia lyase from Fritillaria unibracteata promotes drought tolerance by regulating lignin biosynthesis and SA signaling pathway.

Drought is one of the key threatening environmental factors for plant and agriculture. Phenylalanine ammonia lyase (PAL) is a key enzyme involved in plant defense against abiotic stress, however, the role of PAL in drought tolerance remains elusive. Here, a PAL member (FuPAL1) containing noncanonical Ala-Ser-Gly triad was isolated from Fritillaria unibracteata, one important alpine pharmaceutical plant. FuPAL1, mainly distributed in cytosol, was more conserved than FuCOMT and FuCHI at both nucleotide and amino acid levels. FuPAL1 was overexpressed in Escherichia coli and the purified recombinant FuPAL1 protein showed catalytic preference on L-Phe than L-Tyr. Homology modeling and site-mutation of FuPAL1 exhibited FuPAL1 took part in the ammonization process by forming MIO-like group, and Phe141, Ser208, Ileu218 and Glu490 played key roles in substrate binding and (or) catalysis. HPLC analysis showed that lignin and salicylic acid levels increased but total flavonoid levels decreased in FuPAL1 transgenic Arabidopsis compared to wild-type plants. Moreover, FuPAL1 transgenic Arabidopsis significantly enhanced its drought tolerance, which suggested that FuPAL1 mediated tolerance to drought by inducing the biosynthesis and accumulation of salicylic acid and lignin. Taken together, our results confirmed that the FuPAL1 played an important role in drought tolerance, and FuPAL1 might be a valuable target for genetic improvement of drought resistance in future.

Biochemical Characterization of Novel Phenylalanine Ammonia-Lyase from Spirulina CPCC-695.

Phenylalanine ammonia lyase (PAL) catalyzes the deamination of phenylalanine to cinnamic acid and ammonia. It plays a crucial role in the formation of secondary metabolites through the phenylpropanoid pathway. Recently there has been growing interest in exploring the biochemical properties of PAL for its clinical and commercial applications. PAL as a key component has been used in metabolic engineering and synthetic biology. Due to its high substrate specificity and catalytic efficacy, PAL has opened a new area of interest in the biomedical field. PAL has been frequently used in the enzyme replacement therapy of phenylketonuria, cancer treatment and microbial production of l-phe the precursor of noncalorific sweetener aspartame (Methyl L-α-aspartyl-l-phenylalaninate), antimicrobial and health supplements. PAL occurs in few plants, fungi, bacteria, and cyanobacteria. The present investigation is a preliminary study in which an attempt has been made for the isolation, partial purification, and biochemical characterization of PAL (crude and partially purified) from Spirulina CPCC-695. Partially purified PAL exhibited higher enzymatic activity and protein content than the crude enzyme. Molecular weight of the crude and partially purified PAL was ~ 66 kDa. The optimum temperature and pH for PAL activity was observed as 30 â„ƒ and 8.0 respectively. l-Phe was the most preferred substrate (100 mM) whereas gallic acid showed maximum inhibition of PAL activity. Enzyme kinetics suggested good catalytic efficacy of the PAL enzyme and affinity towards substrate. Both the enzyme (crude and partially purified) showed less than 5% haemolysis suggesting the biocompatible nature of PAL.

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis.

The ginsenoside Rg3 found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg3 in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg3 from Rh2 . Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg3 . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg3 accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg3 accumulation. Finally, we produced ginsenoside Rg3 at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg3 commercially for pharmaceutical use.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Production of trans-cinnamic acid by whole-cell bioconversion from L-phenylalanine in engineered Corynebacterium glutamicum.

BACKGROUND: trans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of L-phenylalanine (L-Phe) into t-CA and developed a repeated bioconversion process. RESULTS: An expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of L-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of L-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation. CONCLUSIONS: We developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

SmKFB5 protein regulates phenolic acid biosynthesis by controlling the degradation of phenylalanine ammonia-lyase in Salvia miltiorrhiza.

Phenolic acids are the major secondary metabolites and significant bioactive constituents of the medicinal plant Salvia miltiorrhiza. Many enzyme-encoding genes and transcription factors involved in the biosynthesis of phenolic acids have been identified, but the underlying post-translational regulatory mechanisms are poorly understood. Here, we demonstrate that the S. miltiorrhiza Kelch repeat F-box protein SmKFB5 physically interacts with three phenylalanine ammonia-lyase (PAL) isozymes and mediates their proteolytic turnover via the ubiquitin-26S proteasome pathway. Disturbing the expression of SmKFB5 reciprocally affected the abundance of SmPAL protein and the accumulation of phenolic acids, suggesting that SmKFB5 is a post-translational regulator responsible for the turnover of PAL and negatively controlling phenolic acids. Furthermore, we discovered that treatment of the hairy root of S. miltiorrhiza with methyl jasmonate suppressed the expression of SmKFB5 while inducing the transcription of SmPAL1 and SmPAL3. These data suggested that methyl jasmonate consolidated both transcriptional and post-translational regulation mechanisms to enhance phenolic acid biosynthesis. Taken together, our results provide insights into the molecular mechanisms by which SmKFB5 mediates the regulation of phenolic acid biosynthesis by jasmonic acid, and suggest valuable targets for plant breeders in tailoring new cultivars.

Aluminum oxide nanoparticles affect the cell wall structure and lignin composition slightly altering the soybean growth.

Aluminum oxide (Al2O3) nanoparticles (NPs) are among the nanoparticles most used industrially, but their impacts on living organisms are widely unknown. We evaluated the effects of 50-1000 mg L-1 Al2O3 NPs on the growth, metabolism of lignin and its monomeric composition in soybean plants. Al2O3 NPs did not affect the length of roots and stems. However, at the microscopic level, Al2O3 NPs altered the root surface inducing the formation of cracks near to root apexes and damage to the root cap. The results suggest that Al2O3 NPs were internalized and accumulated into the cytosol and cell wall of roots, probably interacting with organelles such as mitochondria. At the metabolic level, Al2O3 NPs increased soluble and cell wall-bound peroxidase activities in roots and stems but reduced phenylalanine ammonia-lyase activity in stems. Increased lignin contents were also detected in roots and stems. The Al2O3 NPs increased the p-hydroxyphenyl monomer levels in stems but reduced them in roots. The total phenolic content increased in roots and stems; cell wall-esterified p-coumaric and ferulic acids increased in roots, while the content of p-coumaric acid decreased in stems. In roots, the content of ionic aluminum (Al+3) was extremely low, corresponding to 0.0000252% of the aluminum applied in the nanoparticulate form. This finding suggests that all adverse effects observed were due to the Al2O3 NPs only. Altogether, these findings suggest that the structure and properties of the soybean cell wall were altered by the Al2O3 NPs, probably to reduce its uptake and phytotoxicity.

Biomedical applications of microbial phenylalanine ammonia lyase: Current status and future prospects.

Phenylalanine ammonia lyase (PAL) has recently emerged as an important therapeutic enzyme with several biomedical applications. The enzyme catabolizes l-phenylalanine to trans-cinnamate and ammonia. PAL is widely distributed in higher plants, some algae, ferns, and microorganisms, but absent in animals. Although microbial PAL has been extensively exploited in the past for producing industrially important metabolites, its high substrate specificity and catalytic efficacy lately spurred interest in its biomedical applications. PEG-PAL drug named Palynziq™, isolated from Anabaena variabilis has been recently approved for the treatment of adult phenylketonuria (PKU) patients. Further, it has exhibited high potency in regressing tumors and treating tyrosine related metabolic abnormalities like tyrosinemia. Several therapeutically valuable metabolites have been biosynthesized via its catalytic action including dietary supplements, antimicrobial peptides, aspartame, amino-acids, and their derivatives. This review focuses on all the prospective biomedical applications of PAL. It also provides an overview of the structure, production parameters, and various strategies to improve the therapeutic potential of this enzyme. Engineered PAL with improved pharmacodynamic and pharmacokinetic properties will further establish this enzyme as a highly efficient biological drug.

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.