Vanillin production using metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> under non-growing conditions

Background  

Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details.     

DNA protective properties of vanillin against γ-radiation under different conditions: Possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of γ-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to γ-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of γ-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, ‘tail moment’ and ‘Olive tail moment’. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to γ-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS⁺ radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against γ-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The ability to synthesize chiral building block molecules with high optical purity is of considerable importance to the fine chemical and pharmaceutical industries. Production of one such compound, 3-hydroxyvalerate (3HV), has previously been studied with respect to the in vivo or in vitro enzymatic depolymerization of biologically-derived co-polymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). However, production of this biopolymeric precursor typically necessitates the supplementation of a secondary carbon source (e.g., propionate) into the culture medium. In addition, previous approaches for producing 3HV have not focused on its enantiopure synthesis, and thus suffer from increased costs for product purification.     

Bioproduction of vanillin using an organic solvent-tolerant <Emphasis Type="Italic">Brevibacillus agri</Emphasis> 13

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).     

Crystal structures of <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> dihydropteroate synthase in the apo-form and complexed with the product 7,8-dihydropteroate

Background  

The enzyme dihydropteroate synthase (DHPS) participates in the de novo synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of p-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium Burkholderia cenocepacia is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against Burkholderia and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery.     

Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> to nickel challenge

Background  

Exposure to nickel (Ni) and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress.     

A comparative study of protein synthesis in <Emphasis Type="Italic">in vitro</Emphasis> systems: from the prokaryotic reconstituted to the eukaryotic extract-based

Background  

Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.     

Viable nonsense mutants for the essential gene <Emphasis Type="Italic">SUP45</Emphasis> of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.     

Overexpression of the riboflavin biosynthetic pathway in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes.     

Genome-wide screening of the genes required for tolerance to vanillin,which is a potential inhibitor of bioethanol fermentation,in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Lignocellulosic materials are abundant and among the most important potential sources for bioethanol production. Although the pretreatment of lignocellulose is necessary for efficient saccharification and fermentation, numerous by-products, including furan derivatives, weak acids, and phenolic compounds, are generated in the pretreatment step. Many of these components inhibit the growth and fermentation of yeast. In particular, vanillin is one of the most effective inhibitors in lignocellulose hydrolysates because it inhibits fermentation at very low concentrations. To identify the genes required for tolerance to vanillin, we screened a set of diploid yeast deletion mutants, which are powerful tools for clarifying the function of particular genes.     

Metabolic engineering for the production of shikimic acid in an evolved <Emphasis Type="Italic">Escherichia coli</Emphasis> strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS^-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG ^fbr, tktA, aroB and aroE, on SA synthesis.     

Characterization of a multifunctional methyltransferase from the orchid <Emphasis Type="Italic">Vanilla planifolia</Emphasis>

The final enzymatic step in the synthesis of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) is believed to be methylation of 3,4-dihydroxybenzaldehyde. We have isolated and functionally characterized a cDNA that encodes a multifunctional methyltransferase from Vanilla planifolia tissue cultures that can catalyze the conversion of 3,4-dihydroxybenzaldehyde to vanillin, although 3,4-dihydroxybenzaldehyde is not the preferred substrate. The higher catalytic efficiency of the purified recombinant enzyme with the substrates caffeoyl aldehyde and 5-OH-coniferaldehyde, and its tissue distribution, suggest this methyltransferase may primarily function in lignin biosynthesis. However, since the enzyme characterized here does have 3,4-dihydroxybenzaldehyde-O-methyltransferase activity, it may be useful in engineering strategies for the synthesis of natural vanillin from alternate sources.Abbreviations COMT Caffeic acid O-methyltransferase - DOMT 3,4-Dihydroxybenzaldehyde-O-methyltransferase - OMTs O-Methyltransferases - SAM S-adenosyl-l-methionine     

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.     

Synthesis of chlorophyll <Emphasis Type="Italic">b</Emphasis>: Localization of chlorophyllide <Emphasis Type="Italic">a</Emphasis>oxygenase and discovery of a stable radical in the catalytic subunit

Background  

Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly.     

Melanin in <Emphasis Type="Italic">Fonsecaea pedrosoi</Emphasis>: a trap for oxidative radicals

Background  

The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.     

Synthesis of <Emphasis Type="SmallCaps">L</Emphasis>-ascorbic acid in the phloem

Background  

Although plants are the main source of vitamin C in the human diet, we still have a limited understanding of how plants synthesise L-ascorbic acid (AsA) and what regulates its concentration in different plant tissues. In particular, the enormous variability in the vitamin C content of storage organs from different plants remains unexplained. Possible sources of AsA in plant storage organs include in situ synthesis and long-distance transport of AsA synthesised in other tissues via the phloem. In this paper we examine a third possibility, that of synthesis within the phloem.     

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in <Emphasis Type="Italic">Cynara cardunculus</Emphasis> L

Background  

Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.     

Exobiopolymer production of <Emphasis Type="Italic">Ophiocordyceps dipterigena</Emphasis>BCC 2073: optimization,production in bioreactor and characterization

Background  

Biopolymers have various applications in medicine, food and petroleum industries. The ascomycetous fungus Ophiocordyceps dipterigena BCC 2073 produces an exobiopolymer, a (1→3)-β- D -glucan, in low quantity under screening conditions. Optimization of O. dipterigena BCC 2073 exobiopolymer production using experimental designs, a scale-up in 5 liter bioreactor, analysis of molecular weight at different cultivation times, and levels of induction of interleukin-8 synthesis are described in this study.     

Inactivation of <Emphasis Type="Italic">Dicer1</Emphasis> in Steroidogenic factor 1-positive cells reveals tissue-specific requirement for <Emphasis Type="Italic">Dicer1</Emphasis>in adrenal,testis, and ovary

Background  

The synthesis of microRNA (miRNA) is a multi-step process that requires the action of the ribonuclease Dicer1. Dicer1 is responsible for the final processing of miRNA and has been implicated in cellular processes such as proliferation, apoptosis, and differentiation. Mouse embryos lacking Dicer1 die in early embryogenesis. In this study, we investigated whether Dicer1 is required for development of adrenal, testis, and ovary in mouse embryos.     

Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase,isolated from <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Background  

Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism.