Benzoquinones of the Beetles,Tribolium Castaneum Tribolium Confusum

Abstract

Tribolium castaneum T. confusum were washed in HPLC-grade methanol, and the methanolic washes were analyzed by UV spectroscopy, reversed phase HPLC, and GC/MS. The methanolic washes from both species contained methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ). The amounts of MBQ recovered from the two species were not significantly different, but the amounts of EBQ and total benzoquinones (MBQ + EBQ) recovered from T. castaneum were significantly greater than for those recovered from T. confusum. The methods described are superior to previous methods for isolating, identifying, and quantifying the benzoquinones in these beetles, since they are relatively simple, fast, do not require handling of the beetles, and are sensitive enough to quantify the benzoquinones of a single beetle.     

Benzoquinone levels as a function of age and gender of the red flour beetle, Tribolium castaneum

Liquid chromatography with both photodiode array and electrochemical detection was used to analyze as a function of age and gender the levels of two ρ-quinones, methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ), which are found in defensive secretions of the red flour beetle, Tribolium castaneum. We developed a method to simultaneously analyze quinones and hydroquinones excreted from or in homogenates of individual beetles. The major components present in beetle extracts were the benzoquinones and not free or conjugated forms of the hydroquinones. Greater than 95% of the quinone/hydroquinone mixture in extracts was present in the oxidized form. Because of their lability, however, the quinones were quantified indirectly as their hydroquinone derivatives after extraction in dilute acid supplemented with ascorbic acid as a reducing agent. Comparisons of whole body rinses and homogenates revealed that rinses recovered only up to 60% of the total quinones that were extracted after homogenization. The levels recovered also depended on the age and sex of the individual beetles sampled. ρ-Benzoquinones in both male and female beetles increased after adult eclosion and cuticle sclerotization for 40–50 days and then remained at their highest levels (15–21 μg MBQ and 22–32 μg EBQ per beetle) through 80 days posteclosion. Virgin females that were collected 40–80 days after eclosion contained approximately 40% more of these compounds than males of the same age. The build-up of ρ-benzoquinones subsequent to cuticle sclerotization apparently reflects the need for an adequate cuticular barrier for self-protection from these defensive compounds.     

Volatile secretions and epicuticular hydrocarbons of the beetle Ulomoides dermestoides

Many species of tenebrionids produce and secrete a defensive volatile blend containing mainly benzoquinones and alkenes. In this study we characterized the volatile organic compounds (VOC) of the beetle Ulomoides dermestoides (Coleoptera: Tenebrionidae). Solid phase microextraction (SPME) coupled to capillary gas chromatography–mass spectrometry (CGC–MS) analysis was used to identify methyl-1,4-benzoquinone (MBQ), ethyl-1,4-benzoquinone (EBQ), 1-tridecene (C_13:1), and 1-pentadecene (C_15:1), representing more than 90% of the volatile blend. We also used CGC–MS to analyze the epicuticular hydrocarbons of U. dermestoides. Saturated, unsaturated, and branched structures with chain lengths ranging from 13 to 43 carbons were detected. n-pentacosane (C_25:0) and 9,11-pentacosadiene (9,11-C_25:2) were the most abundant components, representing more than 40% of the cuticular hydrocarbons.     

黄粉虫防御性分泌物抑菌活性的研究

目的:研究黄粉虫防御性分泌物的化学成分及其抑菌活性。方法:用二氯甲烷萃取分泌物并经GC/MS分析,后用牛津杯法和平板连续稀释法分别就该分泌物对大肠杆菌、金黄色葡萄球菌、白色假丝酵母、黑曲霉、桔青霉的抑菌作用及2-甲基对苯醌和对甲酚标准品对上述供试菌的最低抑菌浓度进行测定和比较。结果:分泌物含2-甲基对苯醌、对甲酚和正二十三烷等7种成分,抑菌强度:桔青霉>大肠杆菌和金黄色葡萄球菌>白色假丝酵母>黑曲霉。结论:该分泌物对这5种供试菌的抑制效果优于2-甲基对苯醌和对甲酚标准品。     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.     

An oxidative stress-specific bacterial cell array chip for toxicity analysis

An oxidative stress-specific bacterial cell array chip was fabricated and implemented in the analysis of various different chemicals. The chip consisted of twelve toxicity responsive strains that respond specifically to different oxidative toxicities such as the generation of the superoxide radical, except for strain EBMalK, which was included as a negative control. Each bioluminescent strain carried a fusion of a stress gene promoter (sodA, pqi-5, soxR, fumC, soxS, inaA, hmp, malK, katG, zwf, fpr or pgi) to the bacterial lux reporter genes. A total of nine chemicals were selected to exhibit the capabilities of this array when analyzing different oxidative toxicities. Each of the chemicals were categorized according to their structure and their ability to form radicals in vivo: (I) paraquat, an active radical producer, (II) structural analogs of paraquat that produce radicals, (III) chemicals that are distinct from paraquat but still produce radicals and (IV) chemicals having similar structures as paraquat but do not produce radicals. The results found that each strain was responsive to one or more of the compounds tested but, as a definitive factor, the responses from the chip were dependent upon the production of radicals, i.e., the strains were unresponsive to compounds that were similar in structure to paraquat but lacked the ability to generate radicals. The specificity of the strains used in the chip was also demonstrated by their ability to discriminate between the superoxide radical and hydrogen peroxide. Therefore, this cell array chip could be implemented in characterizing and understanding the toxic impacts of newly synthesized chemicals and drugs in terms of toxicity classification and the nature of oxidative damage experienced by cells.     

Molecular evidence for single Wolbachia infections among geographic strains of the flour beetle Tribolium confusum.

Infections with the rickettsial microorganism Wolbachia are cytoplasmically inherited and occur in a wide range of insect species and several other arthropods. Wolbachia infection often results in unidirectional cytoplasmic incompatibility (CI): crosses between infected males and uninfected females are incompatible and show a reduction of progeny or complete inviability. Unidirectional CI can also occur when males harbouring two incompatible Wolbachia strains are crossed with females infected with only one of the two strains. In the flour beetle Tribolium confusum, Wolbachia infections are of particular interest because of the severity of incompatibility. Typically, no progeny results from the incompatible cross, whereas only partial incompatibility is observed in most other hosts. Werren et al. (1995a) reported that Wolbachia infections in T. confusum consist of two bacterial strains belonging to distinct phylogenic groups, based on PCR amplification and sequence analysis of the bacterial cell division gene ftsZ. However, Fialho & Stevens (1996) showed that eight strains of T. confusum were infected with a single and common incompatibility type. Here we report analysis of the ftsZ gene by specific PCR amplification. Diagnostic restriction enzyme assays revealed no evidence of double infections in 11 geographic strains of T. confusum, including the strain examined by Werren et al. (1995a). Further, sequence analysis of the Wolbachia ftsZ gene and an internal transcribed spacer (ITS) region in two of these strains displayed no nucleotide variation or evidence of polymorphisms. Results suggest that T. confusum is infected with B-group Wolbachia only.     

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high‐value chemicals and next‐generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO₂, H₂O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357–1371, 2016     

产顺,顺-粘康酸细胞工厂的构建与优化

顺,顺-粘康酸是重要的平台化学品。目前,生物合成顺,顺-粘康酸还缺乏高性能菌株,已报道的主要工程菌株不仅需要诱导表达,遗传不稳定,而且发酵培养基组分复杂,不利于大规模工业化生产。构建能利用简单无机盐培养基、遗传稳定且不需要诱导表达的新型工程菌受到人们的关注。本研究在实验室前期构建的产三脱氢莽草酸工程菌株WJ060中,整合合成顺,顺-粘康酸的3个外源基因(aro Z、aro Y、cat A),并且利用3个不同强度的组成型启动子进行组合调控,成功构建了27株顺,顺-粘康酸工程菌,得到的最优工程菌MA30的产量达到1.7 g/L。为了进一步提高顺,顺-粘康酸工程菌的生产能力,利用基因组复制工程构建突变体库,结合高通量筛选方法,经过两轮筛选,成功筛选到了顺,顺-粘康酸产量提高超过8%的大肠杆菌MA30-G2。利用5 L发酵罐进行分批补料发酵,MA30-G2的顺,顺-粘康酸产量达到了11.5 g/L。本研究采用组合调控和高通量筛选相结合的策略不仅促进了顺,顺-粘康酸的生物合成,同时也为其他生物基化学品的生物制造提供了重要参考。     

Generation of an atlas for commodity chemical production in Escherichia coli and a novel pathway prediction algorithm,GEM-Path

The production of 75% of the current drug molecules and 35% of all chemicals could be achieved through bioprocessing (Arundel and Sawaya, 2009). To accelerate the transition from a petroleum-based chemical industry to a sustainable bio-based industry, systems metabolic engineering has emerged to computationally design metabolic pathways for chemical production. Although algorithms able to provide specific metabolic interventions and heterologous production pathways are available, a systematic analysis for all possible production routes to commodity chemicals in Escherichia coli is lacking. Furthermore, a pathway prediction algorithm that combines direct integration of genome-scale models at each step of the search to reduce the search space does not exist. Previous work (Feist et al., 2010) performed a model-driven evaluation of the growth-coupled production potential for E. coli to produce multiple native compounds from different feedstocks. In this study, we extended this analysis for non-native compounds by using an integrated approach through heterologous pathway integration and growth-coupled metabolite production design. In addition to integration with genome-scale model integration, the GEM-Path algorithm developed in this work also contains a novel approach to address reaction promiscuity. In total, 245 unique synthetic pathways for 20 large volume compounds were predicted. Host metabolism with these synthetic pathways was then analyzed for feasible growth-coupled production and designs could be identified for 1271 of the 6615 conditions evaluated. This study characterizes the potential for E. coli to produce commodity chemicals, and outlines a generic strain design workflow to design production strains.     

A comparison study on ribonuclease A modifications induced by substituted p-benzoquinones

In this paper, we present our investigation on ribonuclease A (RNase) modifications induced by 1,4-benzoquinone (PBQ), 2-methyl-1,4-benzoquinone (MBQ), and 2-chloro-1,4-benzoquinone (CBQ). The goal of the study was to evaluate quinone-induced protein modifications as well as substituent effects, utilizing several techniques such as SDS–PAGE, fluorescence spectroscopy, microscopy, and LC-ESI⁺-QTOF-MS. SDS–PAGE experiments revealed that all quinones modify RNase through oligomerization as well as polymeric aggregation; with CBQ functioning as the most efficient quinone while MBQ was least efficient. The fluorescence emission was found to be less intense and the anisotropy values were found to be slightly higher for the modified RNase compared to the unmodified RNase. UV–Vis spectroscopy indicated that all three quinones formed adducts in which they were covalently linked to RNase. Confocal imaging analysis showed that the presence of CBQ resulted in massive RNase aggregation, while PBQ-treated RNase formed much smaller aggregates. MBQ-treated RNase exhibited micrographic features that closely resembled those of the unmodified RNase. LC-ESI⁺-QTOF-MS studies indicated the nature of PBQ- and CBQ-induced RNase modifications are complex mainly due to simultaneously occurrence of both adduct formation and oligomerization. Kinetic studies on quinone reactivity toward lysine revealed the rank order of CBQ > PBQ ≫ MBQ, based on the second-order rate constants. We also utilized scanning electron microscopy in order to investigate the effect of modified RNase on the biomineralization of salts.     

Microbial antagonism: a neglected avenue of natural products research

Competition amongst microbes for space and nutrients in the marine environment is a powerful selective force which has led to the evolution of a variety of effective strategies for colonising and growing on surfaces. We are particularly interested in the chemical ecology of marine epibiotic bacteria which live on the surfaces of marine algae or invertebrates. Over 400 strains of surface-associated bacteria from various species of seaweed and invertebrate from Scottish coastal waters were isolated and 35% of them shown to produce antimicrobial compounds. This is a much higher proportion than free living marine isolates or soil bacteria. In addition, many strains which did not normally produce antibiotics could be induced to do so by exposing them to small amounts of live cells, supernatants from other bacterial cultures or other chemicals. Thus the number of strains able to produce antibiotics appears to be much higher than previously thought. Induction of antibiotic production was elicited by other marine epibionts and also by terrestrial human pathogens such as Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. An understanding of this type of chemical induction and the factors regulating non-constitutive secretion of antimicrobial compounds will allow more effective strategies for searching for new chemotherapeutic antibiotics to be designed.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

Dynamic control in metabolic engineering: Theories,tools, and applications

Metabolic engineering has allowed the production of a diverse number of valuable chemicals using microbial organisms. Many biological challenges for improving bio-production exist which limit performance and slow the commercialization of metabolically engineered systems. Dynamic metabolic engineering is a rapidly developing field that seeks to address these challenges through the design of genetically encoded metabolic control systems which allow cells to autonomously adjust their flux in response to their external and internal metabolic state. This review first discusses theoretical works which provide mechanistic insights and design choices for dynamic control systems including two-stage, continuous, and population behavior control strategies. Next, we summarize molecular mechanisms for various sensors and actuators which enable dynamic metabolic control in microbial systems. Finally, important applications of dynamic control to the production of several metabolite products are highlighted, including fatty acids, aromatics, and terpene compounds. Altogether, this review provides a comprehensive overview of the progress, advances, and prospects in the design of dynamic control systems for improved titer, rate, and yield metrics in metabolic engineering.     

Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production.     

Protein-ubiquinone interaction in bovine heart mitochondrial succinate-cytochrome c reductase. Synthesis and biological properties of fluorine substituted ubiquinone derivatives.

To investigate the protein-ubiquinone interaction in the bovine heart mitochondrial succinate-cytochrome c reductase region of the respiratory chain, three fluorine substituted ubiquinone derivatives, 2,3-dimethoxy-6-(9'-fluorodecyl)-1,4-benzoquinone (9FQ), 2-methoxy-5-trifluoromethyl-6-decyl-1,4-benzoquinone (TFQ), and 2-methoxy-5-trifluoromethyl-6-(9'-fluorodecyl)-1,4-benzoquinone (9FTFQ), were synthesized. 9FQ was synthesized by radical coupling of Q0 and bis(10-fluoroundecanoyl)peroxide. The latter was prepared by fluorination of undecylenic acid followed by thionylchloride treatment and peroxidation. TFQ was synthesized from 2,2,2-trifluoro-p-cresol by methylation, nitration, reduction, acetylation, nitration, reduction, oxidation, and radical alkylation. 9FTFQ was prepared by the radical alkylation of 2-methoxy-5-trifluoromethyl-1,4-benzoquinone with bis(10-fluoroundecanoyl)peroxide. All three fluoro-Q derivatives are active (greater than 50% the activity of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) when used as electron acceptors for succinate-ubiquinone reductase. However, only 9FQ is active when used as an electron donor for ubiquinol-cytochrome c reductase or as an electron mediator for succinate-cytochrome c reductase. Both TFQ and 9FTFQ are competitive inhibitors for ubiquinol-cytochrome c reductase. A 19FNMR peak-broadening effect was observed for 9FQ when it was reconstituted with ubiquinone-depleted ubiquinol-cytochrome c reductase. A drastic up-field chemical shift was observed for TFQ when it was reconstituted with ubiquinone-depleted reductase. These results indicate that the binding environments of the benzoquinone ring and the alkyl side chain of the Q molecule are different. The strong up-field chemical shift for TFQ, and lack of significant chemical shift for 9FQ, suggest that the benzoquinone ring is bound near the paramagnetic cytochrome b heme.     

Distribution of dTDP-glucose-4,6-dehydratase gene and diversity of potential glycosylated natural products in marine sediment-derived bacteria

To investigate the distribution of dTDP-glucose-4,6-dehydratase (dTGD) gene and diversity of the potential 6-deoxyhexose (6DOH) glycosylated compounds in marine microorganisms, a total of 91 marine sediment-derived bacteria, representing 48 operational taxonomic units and belonging to 25 genera, were screened by polymerase chain reaction. In total, 84% of the strains were dTGD gene positive, suggesting 6DOH biosynthetic pathway is widespread in these marine sediment-derived bacteria. BLASTp results of dTGD gene fragments indicate a high chemical diversity of the potential 6DOH glycosylated compounds. Close phylogenetic relationship occurred between dTGDs involved in the production of same or similar 6DOH glycosylated compounds, suggesting dTGD can be used to predict the structure of potential 6DOH glycosylated compounds produced by new strains. In two cases, where dTGD shared ≥85% amino acid identity and close phylogenetic relationship with their counterparts, 6DOH glycosylated compounds were accurately predicted. Our results demonstrate that phylogenetic analysis of dTGD gene is useful for structure prediction of glycosylated compounds from newly isolated strains and can therefore guide the chemical purification and structure identification process. The rapid identification of strains that possess dTGD gene provides a bioinformatics assessment of the greatest potential to produce glycosylated compounds despite the absence of fully biosynthetic pathways or genome sequences.     

Benzoquinone derivatives of Myrsine africana and Maesa lanceolata

The fruits of Myrsine africana afforded two new benzoquinone derivatives, methylvilangin and methylanhydrovilangin. On the other hand, from the fruits of Maesa lanceolata two more novel compounds; 2,5-dihydroxy-3-(nonadec-14-enyl)-benzoquinone and lanciaquinone were isolated. Their structural elucidation was achieved by spectroscopic measurements including 2D NMR experiments.     

X-linked and autosomal inheritance patterns of homologous genes in two species ofTribolium

The tenebrionid beetles Tribolium castaneum and T. confusum are representative of two distinct species groups within their genus. It has been suggested [Smith, S.G. (1952). J. Morphol. 91:325] that the 8AA + neo-XY karyotype of T. confusum was derived from the ancestral 9AA + XY formula, still present in T. castaneum, via the fusion of one pair of autosomes with the X and Y chromosomes during the early divergence of the confusum and castaneum species groups. In the present paper, electrophoretic variation in malic enzyme and hexokinase-1, detected in laboratory strains in Tribolium, is described. Evidence is presented that the genes encoding variation in both enzymes are autosomal in T. castaneum but are X linked in T. confusum. These species-specific patterns of inheritance of homologous gene loci are consistent with the hypothesized karyotypic history of the genus.     

Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges

To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae , to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.