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1.
J Agric Food Chem ; 68(6): 1684-1690, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31957431

RESUMO

The carotenoid-derived volatile ß-ionone plays an important role in the formation of green and black tea flavors due to its low odor threshold, but its formation and the gene(s) involved in its biosynthesis during the tea withering process is(are) still unknown. In this study, we found that the content of ß-ionone increased during the tea withering process catalyzed by an unknown enzyme(s). Correlation analysis of expression patterns of Camellia sinensis carotenoid cleavage dioxygenase genes (CsCCDs) and the ß-ionone content during the withering period revealed CsCCD4 as the most promising candidate. The full-length CsCCD4 gene was amplified from C. sinensis, and the biochemical function of the recombinant CsCCD4 protein was studied after coexpression in Escherichia coli strains engineered to accumulate ß-carotene. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' double bonds. Volatile ß-ionone was detected as the main product by gas and liquid chromatography-mass spectrometry. The accumulation of ß-ionone was consistent with the expression levels of CsCCD4 in different tissues and during the withering process. The CsCCD4 expression was induced by low temperature and mechanical damage stress but not by dehydration stress. The results demonstrate that CsCCD4 catalyzes the production of ß-ionone in the tea plant and provide insight into its formation mechanism during the withering process.


Assuntos
Camellia sinensis/enzimologia , Carotenoides/metabolismo , Dioxigenases/metabolismo , Norisoprenoides/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Biocatálise , Camellia sinensis/química , Camellia sinensis/genética , Camellia sinensis/metabolismo , Dioxigenases/genética , Manipulação de Alimentos , Espectrometria de Massas , Filogenia , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas/classificação , Plantas/enzimologia , Plantas/genética , Alinhamento de Sequência
2.
Plant Sci ; 290: 110303, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31779913

RESUMO

The physiological roles of the plastidial phosphorylase in starch metabolism of higher plants have been debated for decades. While estimated physiological substrate levels favor a degradative role, genetic evidence indicates that the plastidial phosphorylase (Pho1) plays an essential role in starch initiation and maturation of the starch granule in developing rice grains. The plastidial enzyme contains a unique peptide domain, up to 82 residues in length depending on the plant species, not found in its cytosolic counterpart or glycogen phosphorylases. The role of this extra peptide domain is perplexing, as its complete removal does not significantly affect the in vitro catalytic or enzymatic regulatory properties of rice Pho1. This peptide domain may have a regulatory function as it contains potential phosphorylation sites and, in some plant Pho1s, a PEST motif, a substrate for proteasome-mediated degradation. We discuss the potential roles of Pho1 and its L80 domain in starch biosynthesis and photosynthesis.


Assuntos
Fosforilases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Plastídeos/enzimologia , Plantas/enzimologia , Amido/metabolismo
3.
Food Chem ; 309: 125559, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31679850

RESUMO

Plant cell walls are complex structures that are modified throughout development. They are a major contributor to the properties of plant structure and act as barriers against pathogens. The primary cell walls of plants are composed of polysaccharides and proteins. The polysaccharide fraction is divided into components cellulose, hemicelluloses and pectin, are all modified during fruit ripening. Pectin plays an important role in intercellular adhesion and controlling the porosity of the wall. A large number of pectin degrading enzymes have been characterised from plants and they are involved in numerous aspects of plant development. The role of pectate lyases in plant development has received little attention, probably because they are normally associated with the action of plant pathogenic organisms. However their importance in plant development and ripening is now becoming well established and new information about the role of pectate lyases in plant development forms the focus of this review.


Assuntos
Frutas/enzimologia , Plantas/enzimologia , Polissacarídeo-Liase/metabolismo , Frutas/metabolismo , Frutas/fisiologia , Pectinas/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Plantas/metabolismo
4.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569730

RESUMO

Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10-15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Plantas/enzimologia , Plantas/genética , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/química , Epigenômica/métodos , Genoma de Planta , Meiose , Subunidades Proteicas , Origem de Replicação , Transdução de Sinais , Estresse Fisiológico
5.
Int J Mol Sci ; 20(19)2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31546611

RESUMO

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.


Assuntos
5-Metilcitosina/metabolismo , Desmetilação do DNA , DNA Glicosilases/metabolismo , DNA de Plantas/genética , Plantas/enzimologia , DNA Glicosilases/química , Metilação de DNA , DNA de Plantas/química , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Instabilidade Genômica/genética , Óvulo Vegetal/metabolismo , Pólen/metabolismo , Estresse Fisiológico/genética
6.
Plant Sci ; 286: 49-56, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300141

RESUMO

Progress in the functional biochemical analysis of plant glycosyltransferases (GTs) has been slow because plant GTs are generally membrane proteins, operate as part of larger, multimeric complexes, and utilize a vast complexity of substrate acceptors. Therefore, the field would benefit from development of adequate high throughput expression as well as product detection and characterization techniques. Here we review current approaches to tackle such obstacles and suggest a new path forward: nucleic acid programmable protein arrays (NAPPA) with liquid sample desorption ionization (LS-DESI-MS) mass spectrometry. NAPPA utilizes in vitro transcription and translation to produce epitope-tagged fusion proteins from cloned GT cDNAs. LS-DESI is a soft ionization technique that allows rapid and sensitive MS-based product characterization in situ. Coupling both approaches provides the opportunity to examine individual GT functions as well as protein-protein interactions. Furthermore, advances in automated oligosaccharide synthesis and lipid nanodisc technology should allow testing of plant GT activity in presence of numerous substrate acceptors and lipid environments in a high throughput fashion. Thus, NAPPA-DESI-MS has great potential to make headway in biochemical characterization of the large number of plant GTs.


Assuntos
Parede Celular/química , Ensaios de Triagem em Larga Escala/métodos , Plantas/química , Polissacarídeos/biossíntese , Análise Serial de Proteínas/métodos , Glicosiltransferases/análise , Espectrometria de Massas/métodos , Proteínas de Plantas/análise , Plantas/enzimologia
7.
J Mol Model ; 25(8): 240, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31338614

RESUMO

Biofuels such as γ-valerolactone, bioethanol, and biodiesel are derived from potentially fermentable cellulose and vegetable oils. Plant class C GH9 endoglucanases are CBM49-encompassing hydrolases that cleave the ß (1 → 4) glycosidic linkage of contiguous D-glucopyranose residues of crystalline cellulose. Here, I analyse 3D-homology models of characterised and putative class C enzymes to glean insights into the contribution of the GH9, linker, and CBM49 to the mechanism(s) of crystalline cellulose digestion. Crystalline cellulose may be accommodated in a surface groove which is imperfectly bounded by the GH9_CBM49, GH9_linker, and linker_CBM49 surfaces and thence digested in a solvent accessible subsurface cavity. The physical dimensions and distortions thereof, of the groove, are mediated in part by the bulky side chains of aromatic amino acids that comprise it and may also result in a strained geometry of the bound cellulose polymer. These data along with an almost complete absence of measurable cavities, along with poorly conserved, hydrophobic, and heterogeneous amino acid composition, increased atomic motion of the CBM49_linker junction, and docking experiements with ligands of lower degrees of polymerization suggests a modulatory rather than direct role for CBM49 in catalysis. Crystalline cellulose is the de facto substrate for CBM-containing plant and non-plant GH9 enzymes, a finding supported by exceptional sequence- and structural-homology. However, despite the implied similarity in general acid-base catalysis of crystalline cellulose, this study also highlights qualitative differences in substrate binding and glycosidic bond cleavage amongst class C members. Results presented may aid the development of novel plant-based GH9 endoglucanases that could extract and utilise potential fermentable carbohydrates from biomass. Graphical Abstract Crystalline cellulose digestion by plant class C GH9 endoglucanases - an in silico assessment of function.


Assuntos
Celulose/metabolismo , Glicosídeo Hidrolases/metabolismo , Plantas/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalização , Glicosídeo Hidrolases/química , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise de Componente Principal , Homologia Estrutural de Proteína
8.
Biomolecules ; 9(6)2019 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-31181827

RESUMO

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) is a highly conserved protein complex that influences several signaling and developmental processes. The COP9 signalosome consists of eight subunits, among which two subunits, CSN5 and CSN6, contain an Mpr1/Pad1 N-terminal (MPN) domain and the remaining six subunits contain a proteasome, COP9 signalosome, and initiation factor 3 (PCI) domain. In plants, each MPN subunit is encoded by two genes, which is not the case in other organisms. This review aims to provide in-depth knowledge of each COP9 signalosome subunit, concentrating on genetic analysis of both partial and complete loss-of-function mutants. At the beginning of this review, the role of COP9 signalosome in the hormonal signaling and defense is discussed, whereas later sections deal in detail with the available partial loss-of-function, hypomorphic mutants of each subunit. All available hypomorphic mutants are compared based on their growth response and deneddylation activity.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Meio Ambiente , Reguladores de Crescimento de Planta/metabolismo , Plantas/enzimologia , Complexo do Signalossomo COP9/genética , Mutação , Plantas/genética , Plantas/metabolismo , Transdução de Sinais
9.
Phytopathology ; 109(10): 1741-1750, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31179856

RESUMO

Phytopathogens have evolved mechanisms to utilize host genes (commonly known as susceptibility factors) to promote their pathogenesis. Rhizoctonia solani is a highly destructive fungal pathogen of various plants, including rice. We previously reported rice genes that were differentially regulated during R. solani pathogenesis. In this study, we analyzed the role of tomato homologs of two rice genes, isoflavone reductase (IFR) and alternative NADH:ubiquinone oxidoreductase (NUOR), as potential susceptibility factors for R. solani. Virus-induced gene silencing of NUOR in tomato resulted in compromised susceptibility against R. solani, whereas IFR-silenced plants demonstrated susceptibility similar to that of control plants. NUOR silencing in tomato led to homogenous accumulation of reactive oxygen species (optimum range) upon R. solani infection. In addition, the expression and enzyme activity of some host defense and antioxidant genes was enhanced, whereas H2O2 content, lipid peroxidation, and electrolyte leakage were reduced in NUOR-silenced plants. Similarly, transient silencing of OsNUOR provided tolerance against R. solani infection in rice. Overall, the data presented in this study suggest that NUOR serves as a host susceptibility factor to promote R. solani pathogenesis.


Assuntos
Resistência à Doença , Complexo I de Transporte de Elétrons , Plantas , Rhizoctonia , Resistência à Doença/genética , Complexo I de Transporte de Elétrons/metabolismo , Oryza/enzimologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Plantas/enzimologia , Plantas/microbiologia , Rhizoctonia/fisiologia
10.
Int J Mol Sci ; 20(12)2019 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-31234541

RESUMO

Omega-3 long chain polyunsaturated fatty acids (ω3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3) are important fatty acids for human health. These ω3 LC-PUFAs are produced from their ω3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from ω6 LC-PUFA by omega-3 desaturases (ω3Ds). Here, we have investigated eight ω3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 ω6 PUFA substrates by transiently expressing them in Nicotiana benthamiana leaves. Our results showed hitherto unreported activity of many of the ω3Ds on ω6 LC-PUFA substrates leading to their conversion to ω3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Animais , Cianobactérias/enzimologia , Fungos/enzimologia , Plantas/enzimologia , Plantas Geneticamente Modificadas , Especificidade por Substrato , Tabaco/genética
11.
Mol Plant Microbe Interact ; 32(10): 1378-1390, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31063047

RESUMO

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.


Assuntos
Quitina , Quitinases , Proteínas Fúngicas , Doenças das Plantas , Plantas , Verticillium , Proteínas de Transporte , Quitina/metabolismo , Proteínas Fúngicas/metabolismo , Simulação de Acoplamento Molecular , Doenças das Plantas/microbiologia , Plantas/enzimologia , Plantas/imunologia
12.
Prep Biochem Biotechnol ; 49(8): 744-758, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050587

RESUMO

Lignocellulosic biomass (LB) is the renewable feedstock for the production of fuel/energy, feed/food, chemicals, and materials. LB could also be the versatile source of the functional oligosaccharides, which are non-digestible food ingredients having numerous applications in food, cosmetics, pharmaceutical industries, and others. The burgeoning functional food demand is expected to be more than US$440 billion in 2022. Because of higher stability at low pH and high temperature, oligosaccharides stimulate the growth of prebiotic bifidobacteria and lactic acid bacteria. Xylooligosaccharides (XOS) are major constituents of oligosaccharides consisting of 2-7 xylose monomeric units linked via ß-(1,4)-linkages. XOS can be obtained from various agro-residues by thermochemical pretreatment, enzymatic or chemoenzymatic methods. While thermochemical methods are fast, reproducible, enzymatic methods are substrate specific, costly, and produce minimum side products. Enzymatic methods are preferred for the production of food grade and pharmaceutically important oligosaccharides. XOS are potent prebiotics having antioxidant properties and enhance the bio-adsorption of calcium and improving bowel functions, etc. LB can cater to the increasing demand of oligosaccharides because of their foreseeable amount and the advancements in technology to recover oligosaccharides. This paper summarizes the methods for oligosaccharides production from LB, classification, and benefits of oligosaccharides on human health.


Assuntos
Biotecnologia/métodos , Glucuronatos/metabolismo , Lignina/metabolismo , Oligossacarídeos/metabolismo , Biomassa , Fibras na Dieta/análise , Humanos , Plantas/enzimologia , Plantas/metabolismo , Xilosidases/metabolismo
13.
Adv Exp Med Biol ; 1142: 253-272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31102250

RESUMO

Plant chitinase hydrolyzing ß-1,4-glycosidic linkages of chitin are major enzymes acting in plant-microbe interactions and are involved in self-defense against fungal pathogens. Chitosanases from soil bacteria are also involved in plant defense by hydrolyzing chitosan components of the fungal cell wall. The crystal structures of these enzymes in complex with their substrates have been solved, and the mechanisms of substrate binding were elucidated at the atomic level. These findings enabled us to speculate on the enzyme targets under physiological conditions, leading us to define the physiological roles of the enzymes. The structures and functions of chitin/chitosan-binding modules appended to modular chitinases/chitosanases were analyzed by NMR and isothermal titration calorimetry (ITC), and the enzymes were found to form an appropriate modular organization to fulfill their roles in plant-microbe interactions.


Assuntos
Quitinases , Fungos/patogenicidade , Plantas/enzimologia , Plantas/microbiologia , Parede Celular , Quitina , Quitosana
14.
Prog Lipid Res ; 75: 100987, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31078649

RESUMO

Most current knowledge about plant lipid metabolism has focused on the biosynthesis of lipids and their transport between different organelles. However, lipid composition changes during development and in response to environmental cues often go beyond adjustments of lipid biosynthesis. When lipids have to be removed to adjust the extent of membranes during down regulation of photosynthesis, or lipid composition has to be adjusted to alter the biophysical properties of membranes, or lipid derived chemical signals have to be produced, lipid-degrading enzymes come into play. This review focuses on glycerolipid acylhydrolases that remove acyl groups from glycerolipids and will highlight their roles in lipid remodeling and lipid-derived signal generation. One emerging theme is that these enzymes are involved in the dynamic movement of acyl groups through different lipid pools, for example from polar membrane lipids to neutral lipids sequestered in lipid droplets during de novo triacylglycerol synthesis. Another example of acyl group sequestration in the form of triacylglycerols in lipid droplets is membrane lipid remodeling in response to abiotic stresses. Fatty acids released for membrane lipids can also give rise to potent signaling molecules and acylhydrolases are therefore often the first step in initiating the formation of these lipid signals.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Plantas/enzimologia , Membrana Celular/química , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Proteínas de Plantas/metabolismo
15.
World J Microbiol Biotechnol ; 35(5): 70, 2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31011828

RESUMO

Cyanide is a nitrile which is used extensively in many industries like jewelry, mining, electroplating, plastics, dyes, paints, pharmaceuticals, food processing, and coal coking. Cyanides pose a serious health hazard due to their high affinity towards metals and cause malfunction of cellular respiration by inhibition of cytochrome c oxidase. This inhibition ultimately leads to histotoxic hypoxia, increased acidosis, reduced the functioning of the central nervous system and myocardial activity. Different physicochemical processes including oxidation by hydrogen peroxide, alkaline chlorination, and ozonization have been used to reduce cyanide waste from the environment. Microbial cyanide degradation which is considered as one the most successful techniques is used to take place through different biochemical/metabolic pathways involving reductive, oxidative, hydrolytic or substitution/transfer reactions. Groups of enzymes involved in microbial degradation are cyanidase, cyanide hydratase, formamidase, nitrilase, nitrile hydratase, cyanide dioxygenase, cyanide monooxygenase, cyanase and nitrogenase. In the future, more advancement of omics technologies and protein engineering will help us to recoup the environment from cyanide effluent. In this review, we have discussed the origin and environmental distribution of cyanide waste along with different bioremediation pathways and enzymes involved therein.


Assuntos
Bactérias/enzimologia , Cianetos/metabolismo , Fungos/enzimologia , Resíduos Industriais/análise , Plantas/enzimologia , Amidoidrolases , Aminoidrolases , Bactérias/classificação , Bactérias/metabolismo , Biodegradação Ambiental , Carbono-Nitrogênio Liases , Cianetos/toxicidade , Fungos/metabolismo , Hidroliases , Hidrolases , Microbiologia Industrial , Nitrogenase , Oxirredução , Plantas/classificação , Plantas/metabolismo , Sulfurtransferases , Tiossulfato Sulfurtransferase
17.
Appl Microbiol Biotechnol ; 103(9): 3715-3725, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30915501

RESUMO

Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from p-coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in Escherichia coli. When the RZS1 gene was introduced into E. coli and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3-4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced p-coumaroyl-CoA from p-coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from Arabidopsis thaliana seemed to be more suitable for raspberry ketone production than that from Petroselinum crispum. This work paves an alternative way for future economic production of natural raspberry ketone.


Assuntos
Butanonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Vias Biossintéticas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Propionatos/metabolismo
18.
Planta ; 249(6): 1695-1714, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30895445

RESUMO

MAIN CONCLUSION: This paper provides an overview on activity, stereospecificity, expression and regulation of pinoresinol-lariciresinol reductases in plants. These enzymes are shared by the pathways to all 8-8' lignans derived from pinoresinol. Pinoresinol-lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols. They catalyze two successive reduction steps leading to the production of lariciresinol or secoisolariciresinol from pinoresinol. Two secoisolariciresinol enantiomers can be synthetized with different fates. Depending on the plant species, these enantiomers are either final products (e.g., in the flaxseed where it is stored after glycosylation) or are the starting point for the synthesis of a wide range of lignans, among which the aryltetralin type lignans are used to semisynthesize anticancer drugs such as Etoposide®. Thus, the regulation of the gene expression of PLRs as well as the possible specificities of these reductases for one reduction step or one enantiomer are key factors to fine-tune the lignan synthesis. Results published in the last decade have shed light on the presence of more than one PLR in each plant and revealed various modes of action. Nevertheless, there are not many results published on the PLRs and most of them were obtained in a limited range of species. Indeed, a number of them deal with wild and cultivated flax belonging to the genus Linum. Despite the occurrence of lignans in bryophytes, pteridophytes and monocots, data on PLRs in these taxa are still missing and indeed the whole diversity of PLRs is still unknown. This review summarizes the data, published mainly in the last decade, on the PLR gene expression, enzymatic activity and biological function.


Assuntos
Furanos/metabolismo , Regulação da Expressão Gênica de Plantas , Lignanas/metabolismo , Oxirredutases/metabolismo , Plantas/enzimologia , Butileno Glicóis/metabolismo , Regulação Enzimológica da Expressão Gênica , Oxirredutases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética
19.
Int J Mol Sci ; 20(6)2019 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-30884891

RESUMO

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.


Assuntos
Agricultura , Biotecnologia , Agricultura Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas/genética , Inibidores de Serino Proteinase/genética , Agricultura/métodos , Animais , Biotecnologia/métodos , Agricultura Molecular/métodos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Plantas/enzimologia , Plantas/microbiologia , Plantas/parasitologia , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/microbiologia , Plantas Geneticamente Modificadas/parasitologia , Proteínas Recombinantes/genética
20.
Plant Physiol Biochem ; 136: 245-254, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30710774

RESUMO

Roots of the higher plants can assimilate inorganic nitrogen by an enzymatic reduction of the most oxidized form (+6) nitrate to the reduced form (-2) glutamate. For such reactions, the substrates (originated from photosynthates) must be imported to supply energy through the reductant-generating systems within the root cells. Intensive studies over last 70 years (reviewed here) revealed the precise mechanisms of nitrate-to-glutamate transformation in roots with elaborate searches of 15N-tracing, enzymes involved, the reductant-supplying system, and nitrate signaling. In the 1970s, the tracing of 15N-labeled nitrate and ammonia in the roots demonstrated the sequential reduction and assimilation of nitrate to nitrite, ammonia, glutamine amide, and then glutamate. These reactions involve nitrate reductase (NADH-NR, EC 1.7.1.1) in the cytosol, nitrite reductase (ferredoxin [Fd]-NiR, EC 1.7.7.1), glutamine synthetase (GS2, EC 6.3.1.2), and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in the plastids. NADH for NR is generated by glycolysis in the cytosol, and NADPH for Fd-NIR and Fd-GOGAT are produced by the oxidative pentose phosphate pathway (OPPP). Electrons from NADPH are conveyed to reduce NIR and Fd-GOGAT through Fd-NADP+ reductase (FNR, EC 1.6.7.1) specifically in the roots. Physiological and molecular analyses showed the parallel inductions of NR, NIR, GS2, Fd-GOGAT, OPPP enzymes, FNR, and Fd in response to a short-term nitrate supply. Recent studies proposed a molecular mechanism of nitrate-induction of these genes and proteins. Roots can also assimilate the reduced form of inorganic ammonia by the combination of cytosolic GS1 and plastidic NADH-GOGAT.


Assuntos
Glutamatos/metabolismo , Nitratos/metabolismo , Raízes de Plantas/metabolismo , Plantas/metabolismo , Isótopos de Nitrogênio/metabolismo , Raízes de Plantas/enzimologia , Plantas/enzimologia , Transdução de Sinais
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