Metabolic engineering of heterologous resveratrol production in Saccharomyces cerevisiae faces challenges as the precursor l-tyrosine is stringently regulated by a complex biosynthetic system. We overexpressed the main gene targets in the upstream pathways to investigate their influences on the downstream resveratrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.14±3.74 mg·L–1, 2.27 times higher than the initial production in YPD medium (4% glucose). The newly discovered gene targets TRP1 expressing phosphoribosylanthranilate isomerase, ARO3 expressing 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.
Pyrazole carboxamide derivatives represent an important class of fungicides in agrochemicals. To find more novel structural pyrazole carboxamides, a novel series of 3-(trifluoromethyl)-1H-pyrazole-4-carboxamide compounds were prepared from ethyl 4,4,4-trifluoroacetoacetate and triethyl orthoformate as starting materials. All the products were characterized by Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), 13C NMR, 19F NMR and mass spectrography. The bioassay results showed these fluorine-containing pyrazole carboxamides have a weak fungicidal activity but some of them exhibit a good nematocidal activity against M. incognita.
Pyrroloquinoline quinone (PQQ) plays a significant role as a redox cofactor in combination with dehydrogenases in bacteria. These dehydrogenases play key roles in the oxidation of important substrates for the biotechnology industry, such as vitamin C production. While biosynthesis of PQQ genes has been widely studied, PQQ-transport mechanisms remain unclear. Herein, we used both two-dimensional fluorescence-difference gel electrophoresis tandem mass spectrometry and RNA sequencing to investigate the effects of pqqB overexpression in an industrial strain of Gluconobacter oxydans WSH-003. We have identified 73 differentially expressed proteins and 99 differentially expressed genes, a majority of which are related to oxidation-reduction and transport processes by gene ontology analysis. We also described several putative candidate effectors that responded to increased PQQ levels resulting from pqqB overexpression. Furthermore, quantitative PCR was used to verify five putative PQQ-transport genes among different PQQ producing strains, and the results showed that ompW, B932_1930 and B932_2186 were upregulated in all conditions. Then the three genes were over-expressed in G. oxydans WSH-003 and PQQ production were detected. The results showed that extracellular PQQ of B932_1930 (a transporter) and B932_2186 (an ABC transporter permease) overexpression strains were enhanced by 1.77-fold and 1.67-fold, respectively. The results suggest that the proteins encoded by PqqB, B932_1930 and B932_2186 might enhance the PQQ secretion process.
A robust and versatile tool for multigene pathway assembly is a key to the biosynthesis of high-value chemicals. Here we report the rapid construction of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative toolbox (pUMRI) developed in our research group, which has features of ready-to-use, convenient marker recycling, arbitrary element replacement, shuttle plasmid, auxotrophic marker independence, GAL regulation, and decentralized assembly. Functional isoprenoid biosynthesis pathways containing 4–11 genes with lengths ranging from ~10 to ~22 kb were assembled using this toolbox within 1–5 rounds of reiterative recombination. In combination with GAL-regulated metabolic engineering, high production of isoprenoids (e.g., 16.3 mg?g–1 dcw carotenoids) was achieved. These results demonstrate the wide range of application and the efficiency of the pUMRI toolbox in multigene pathway construction of S. cerevisiae.
A palladium catalyst supported on 2-aminopyridine functionalized cellulose was synthesized and fully characterized by inductively coupled plasma atomic emission spectroscopy, transmission electron microscope, Fourier transform infrared spectroscopy, thermogravimetric analysis and X-ray photoelectron spectrometry. This catalyst can be applied in the Suzuki cross-coupling reaction of aryl halides with arylboronic acids in 50% ethanol to afford biaryls in good yields, and easily recycled by simple filtration after reaction without the loss of metal Pd.
Great interests have arisen over the last decade in the development of hierarchically porous materials. The hierarchical structure enables materials to have maximum structural functions owing to enhanced accessibility and mass transport properties, leading to improved performances in various applications. Hierarchical porous materials are in high demand for applications in catalysis, adsorption, separation, energy and biochemistry. In the present review, recent advances in synthesis routes to hierarchically porous materials are reviewed together with their catalytic contributions.
The effect of thermal pretreatment on the active sites and catalytic performances of PtSn/SiO2 catalyst in acetic acid (AcOH) hydrogenation was investigated in this article. The catalysts were characterized by N2 physical adsorption, X-ray diffraction, transmission electron microscopy, pyridine Fourier-transform infrared spectra, and H2-O2 titration on its physicochemical properties. The results showed that Pt species were formed primarily in crystalline structure and no PtSnx alloy was observed. Meanwhile, with the increment of thermal pretreatment temperature, Pt dispersion showed a decreasing trend due to the aggregation of Pt particles. Simultaneously, the amount of Lewis acid sites was remarkably influenced by such thermal pretreatment owning to the consequent physicochemical property variation of Sn species. Interestingly, the catalytic activity showed the similar variation trend with that of Lewis acid sites, confirming the important roles of Lewis acid sites in AcOH hydrogenation. Moreover, a balancing effect between exposed Pt and Lewis acid sites was obtained, resulting in the superior catalytic performance in AcOH hydrogenation.
Aqueous solutions of methyldiethanolamine (MDEA) and piperazine (PZ) are commonly used solvent nowadays. In this work a thermodynamic analysis with the Electrolyte-NRTL model has been performed for systems composed of acidic gases and MDEA + PZ aqueous solution. ASPEN Plus® has been used for thermodynamic modeling. Values of binary interaction parameters for liquid phase activity coefficients have been estimated from regressions of experimental data. Moreover, the influence of the interactions between ion pairs and MDEA or PZ molecular species has been analyzed. The final aim is to obtain a reliable tool for design and simulation of absorption and stripping columns, fundamentals also in order to carry out energy saving studies.
Biodegradable poly(ether-imide-ester) elastomers were synthesized from succinic acid, 1,4-butanediol, polyethylene glycol 1000 and N′,N-bis(2-carboxyethyl)- pyromellitimide which was derived from pyromellitic dianhydride and glycine. The chemical structures, crystallinities, thermal stabilities, mechanical properties, hydrophilicities and biodegradabilities of these elastomers were investigated. The hard segments of the linear aliphatic poly (ether-ester) exhibited monoclinic chain packing. Increasing the amount of aromatic bisimide moieties in the poly (ether-ester) reduced the crystallinity of the material and improved the thermal stability and tensile strength of the elastomers. In addition, introducing a suitable amount of aromatic bisimide moieties into the poly(ether-ester) backbones endowed the elastomers with improved biodegradability but too many aromatic bisimide groups reduced the biodegradability of the elastomers.
A review of recent research related to microporous polymeric membranes formed via thermally induced phase separation (TIPS) and the morphologies of these membranes is presented. A summary of polymers and suitable diluents that can be used to prepare these microporous membranes via TIPS are summarized. The effects of different kinds of polymer materials, diluent types, cooling conditions, extractants and additive agents on the morphology and performance of TIPS membranes are also discussed. Finally new developments in TIPS technology are summarized.
X-ray crystallography is a powerful strategy for 3-D structure determination of macromolecules, such as nucleic acids and protein-nucleic acid complexes. However, the crystallization and phase determination are the major bottle-neck problems in crystallography. Recently we have successfully developed synthesis and strategy of selenium-derivatized nucleic acids (SeNA) for nucleic acid crystallography. SeNA might not only provide the rational strategies to solve the phase determination problem, but also offer a potential strategy to explore crystallization solutions.
Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein (YFP) reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.
Ribozymes are widespread, and catalyze some extremely important reactions in the cell. Mechanistically most fall into one of two classes, using either metal ions or general acid-base catalysis. The nucleolytic ribozymes fall into the latter class, mostly using nucleobases. A sub-set of these use a combination of guanine base plus adenine acid to catalyze the cleavage reaction. New ribozymes are still being discovered at regular intervals and we can speculate on the potential existence of ribozymes that catalyze chemistry beyond phosphoryl transfer reactions, perhaps using small-molecule coenzymes.
Amyloid peptides are renowned to be related to neurodegenerative diseases, however, a fruitful avenue is to employ them as high-performance nanomaterials. These materials benefit from the intrinsic outstanding mechanical robustness of the amyloid backbone made of β-strands. In this work, we exploited amyloid-like fibrils as functional material to attach pristine L-cysteine aggregates (cystine oligomers) and gold nanoparticles, without the need of templating compounds. This work will open new avenues on functional materials design and their realisation.
129Te(129I) and 119Sn Mössbauer investigations have revealed that the structure of Ge100 ? xSx and Ge100 ? xSex glasses enriched in the chalcogen (x ≥ 0.66) is built up of structural units containing twofold-coordinated chalcogen (X) atoms in chains of the
types, whereas germanium atoms in these glasses have the oxidation state of only +4 and are fourfold-coordinated so that the local environment of the germanium atoms includes only chalcogen atoms. The structure of the glasses depleted in the chalcogen consists of structural units containing twofold-coordinated chalcogen atoms in chains of the
type, whereas germanium in these glasses is stabilized both in the oxidation state of +4 and the fourfold-coordinated state and in the oxidation state of +2 and the threefold-coordinated state so that the local environment of the germanium atoms includes only chalcogen atoms.
Natural products and their derivatives represent a rich source for the discovery and development of new cancer therapeutic drugs. Bioactive components derived from natural sources including marine compounds have been shown to be effective agents in the clinic or in preclinical settings. In the present review, we present a story of discovery, synthesis and evaluation of three synthetic tricyclic pyrroloquinone (TPQ) alkaloid analogs as cancer therapeutic agents. Chemical synthesis of these compounds (BA-TPQ, TBA-TPQ, and TCBA-TPQ) has been accomplished and the mechanisms of action (MOA) and structure-activity relationships (SAR) have been investigated. In the past, the complexity of chemical synthesis and the lack of well-defined MOA have dampened the enthusiasm for the development of some makaluvamines. Recent discovery of novel molecular targets for these alkaloids (unrelated to inhibition of Topoisomerase II) warrant further consideration as clinical candidates in the future. In addition to the establishment of novel synthetic approaches and demonstration of in vitro and in vivo anticancer activities, we have successfully demonstrated that these makaluvamines attack several key molecular targets, including the MDM2-p53 pathway, providing ample opportunities of modulating the compound structure based on SAR and the use of such compounds in combination therapy in the future.
The antioxidant and antitumor effects as well as the immunomodulatory activities of crude and purified polyphenol extract from blueberries were investigated. The antioxidant and antitumor effects of the polyphenol extract were measured both in vitro and in vivo, and their effect on the immune systems of CD-1 tumor-bearing mice were also analyzed. In vitro assays demonstrated that blueberry purified polyphenol extract (BBPP) exhibited higher antioxidant activities than blueberry crude polyphenol extract (BBCP), but the opposite effect was observed in vivo. Both the in vitro and in vivo antitumor activity and the immunity assay showed that BBCP not only inhibited tumor growth, but also significantly improved the immunity of the mice. According to physical and histological studies, the CD-1 tumor-bearing mice treated with the polyphenol extract, especially high doses of BBCP experienced a higher quality of life than the positive control group (treated with cyclophosphamide). These results indicate that BBCP has significant antioxidant and antitumor activities and that it can enhance the immunity of CD-1 tumor-bearing mice.
Rhamnolipids are one of the most effective biosurfactants that are of great interest in industrial applications such as enhancing oil recovery, health care, cosmetics, pharmaceutical processes, food processing, detergents for protein folding, and bioremediation due to their unique characteristics such as low toxicity, surface active property to reduce surface/interfacial tensions, and excellent biodegradability. The genes and metabolic pathways for rhamnolipid synthesis have been well elucidated, but its cost-effective production is still challenging. Pseudomonas aeruginosa, the most powerful rhamnolipid producer, is an opportunistic pathogen, which limits its large scale production and applications. Rhamnolipid production using engineered strains other than Pseudomonas aeruginosa such as E. coli and Pseudomonas putida has received much attention. The highest yield of rhamnolipids is achieved when oil-type carbon sources are used, but using cheaper and renewable carbon sources such as lignocellulose would be an attractive strategy to reduce the production cost of rhamnolipids for various industrial applications.
A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We examined G-quadruplex forming ability of GGGGCCrepeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K+ ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5'- and 3'-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[(G4C2)3G4] was chosen for further scrutiny and optimization of nuclear magnetic resonance (NMR) spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when substituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG substitution at position 21 and fine tuning of folding conditions facilitate folding of d[(G4C2)3GGBrGG] into (mostly) a single G-quadruplex, and thus enable determination of its high-resolution structure by high-field NMR.
Rhamnolipids are a class of biosurfactants that have a great potential to be used in industries. Five proteins/enzymes, namely RhlA, RhlB, RhlC, RhlG and RhlI, are critical for the production of rhamnolipids in Pseudomonas aeruginosa. Four of the 5 proteins except RhlC were successfully over-expressed in E. coli and three of them (RhlA, RhlB and RhlI) were purified and obtained in milligram quantities. The purified proteins were shown to be folded in solution. Homology models were built for RhlA, RhlB and RhlI. These results lay a basis for further structural and functional characterization of these proteins in vitro to favor the construction of super strains for rhamnolipids production.
