Regioselective Hydroxylation of <i>trans</i>-Resveratrol <i>via</i> Inhibition of Tyrosinase from <i>Streptomyces avermitilis</i> MA4680

Secreted tyrosinase from melanin-forming <i>Streptomyces avermitilis</i> MA4680 was involved in both ortho-hydroxylation and further oxidation of <i>trans</i>-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of <i>melC</i>_<i>2</i> and <i>melD</i>_<i>2</i> encoding extracellular and intracellular tyrosinase, respectively; the <i>melC2</i> deletion mutant did not produce piceatannol as well as melanin, whereas the <i>melD2</i> deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently converted <i>trans</i>-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of <i>trans</i>-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of <i>trans</i>-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of <i>trans</i>-resveratrol was dependent upon inhibitor concentration. Using 1.8 mg of wild-type <i>Streptomyces avermitilis</i> cells, the conversion yield of 100 μM <i>trans</i>-resveratrol to piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h^–1 DCW mg^–1 specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases

High-Throughput Quantitative Analysis of Total <i>N</i>-Glycans by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Accurate and reproducible quantification of glycans from protein drugs has become an important issue for quality control of therapeutic proteins in biopharmaceutical and biotechnology industries. Mass spectrometry is a promising tool for both qualitative and quantitative analysis of glycans owing to mass accuracy, efficiency, and reproducibility, but it has been of limited success in quantitative analysis for sialylated glycans in a high-throughput manner. Here, we present a solid-phase permethylation-based total <i>N</i>-glycan quantitative method that includes <i>N</i>-glycan releasing, purification, and derivatization on a 96-well plate platform. The solid-phase neutralization enabled us to perform reliable absolute quantification of the acidic <i>N</i>-glycans as well as neutral <i>N</i>-glycans from model glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin) by only using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, low-abundance sialylated <i>N</i>-glycans from human serum prostate specific antigen (PSA), an extremely valuable prostate cancer marker, were initially quantified, and their chemical compositions were proposed. Taken together, these results demonstrate that our all-inclusive glycan preparation method based on a 96-well plate platform may contribute to the precise and reliable qualitative and quantitative analysis of glycans

The ratio of reaction rate constants of () stem II, stem I and stem I/III modified aptazymes for HCV replicase and () stem II, stem III and stem I/III modified aptazymes at 10 mM Tris–HCl, 100 mM KCl and 10 mM MgCl (pH 7

<p><b>Copyright information:</b></p><p>Taken from "Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal"</p><p>Nucleic Acids Research 2005;33(20):e177-e177.</p><p>Published online 27 Nov 2005</p><p>PMCID:PMC1292994.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>6). The ratio of reaction rate constants means ‘the ratio of the rate constant in the presence of protein to that in the absence of protein’. The concentrations of the aptazymes and substrate in the reaction mixture are 1 µM. Reaction rate constants were obtained from {ln[( − )/]/Δ} ( and are the fluorescence intensities at 5 and at 0 min, respectively). The blank rate constant of stem II mono-aptazyme, stem I mono-aptazyme and stem I/III bis-aptazymes in the absence of HCV replicase were 0.660, 0.757 and 0.254 min, respectively. And the blank rate constants of the stem II mono-aptazyme, stem III mono-aptazyme and stem I/III bis-aptazymes in the absence of HCV helicase (b) were 0.715, 0.782 and 0.019 min, respectively

A Novel Approach for Gene Expression Optimization through Native Promoter and 5′ UTR Combinations Based on RNA-seq, Ribo-seq, and TSS-seq of <i>Streptomyces coelicolor</i>

Streptomycetes are Gram-positive mycelial bacteria, which synthesize a wide range of natural products including over two-thirds of the currently available antibiotics. However, metabolic engineering in <i>Streptomyces</i> species to overproduce a vast of natural products are hampered by a limited number of genetic tools. Here, two promoters and four 5′ UTR sequences showing constant strengths were selected based upon multiomics data sets from <i>Streptomyces coelicolor</i> M145, including RNA-seq, Ribo-seq, and TSS-seq, for controllable transcription and translation. A total eight sets of promoter/5′ UTR combinations, with minimal interferences of promoters on translation, were constructed using the transcription start site information, and evaluated with the GusA system. Expression of GusA could be controlled to various strengths in three different media, in a range of 0.03- to 2.4-fold, compared to that of the control, ermE*P/Shine-Dalgarno sequence. This method was applied to engineer three previously reported promoters to enhance gene expressions. The expressions of ActII-ORF4 and MetK were also tuned for actinorhodin overproductions in <i>S. coelicolor</i> as examples. In summary, we provide a novel approach and tool for optimizations of gene expressions in <i>Streptomyces coelicolor</i>

Additional file 3: of Genome-scale model-driven strain design for dicarboxylic acid production in Yarrowia lipolytica

Biomass composition of Y. lipolytica in C- and N- limited conditions and GAM and NGAM calculations. (DOCX 52 kb

Additional file 2: of Genome-scale model-driven strain design for dicarboxylic acid production in Yarrowia lipolytica

SMBL file of iYLI647. (XML 2312 kb

Additional file 1: of Genome-scale model-driven strain design for dicarboxylic acid production in Yarrowia lipolytica

Changed, added and deleted reactions in iYLI647 model in comparison with iMK735 scaffold model. (XLSX 22 kb

Biosynthesis of (−)-5-Hydroxy-equol and 5‑Hydroxy-dehydroequol from Soy Isoflavone, Genistein Using Microbial Whole Cell Bioconversion

Equols are isoflavandiols formed by reduction of soy isoflavones such as daidzein and genistein by gut microorganisms. These phytoestrogens are of interest for their various biological effects. We report biosynthesis from genistein to (−)-5-hydroxy-equol in recombinant <i>E. coli</i> expressing three reductases (daidzein reductase DZNR, dihidrodaidzein reductase DHDR, tetrahydrodaidzein reductase THDR) and a racemase (dihydrodaidzein racemase, DDRC) originating from the gut bacterium, <i>Slackia isoflavoniconvertens</i>. The biosynthesized 5-hydroxy-equol proved as an optically negative enantiomer, nonetheless it displayed an inverse circular dichroism spectrum to (<i>S</i>)-equol. Compartmentalized expression of DZNR and DDRC in one <i>E. coli</i> strain and DHDR and THDR in another increased the yield to 230 mg/L and the productivity to 38 mg/L/h. If the last reductase was missing, the intermediate spontaneously dehydrated to 5-hydroxy-dehydroequol in up to 99 mg/L yield. This novel isoflavene, previously not known to be synthesized in nature, was also detected in this biotransformation system. Although (<i>S</i>)-equol favors binding to human estrogen receptor (hER) β over hERα, (−)-5-hydroxy-equol showed the opposite preference. This study provides elucidation of the biosynthetic route of (−)-5-hydroxy-equol and measurement of its potent antagonistic character as a phytoestrogen for the first time

Cooperative Catechol-Functionalized Polypept(o)ide Brushes and Ag Nanoparticles for Combination of Protein Resistance and Antimicrobial Activity on Metal Oxide Surfaces

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept­(o)­ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly­(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure–property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections

Development of High Performance Polyurethane Elastomers Using Vanillin-Based Green Polyol Chain Extender Originating from Lignocellulosic Biomass

Vanillin can be obtained from waste of lignocellulosic bioresources with various methods.− Such vanillin was used as chain extender [divanillin-ethanol amine conjugate (DV-EA)] after its dimerization and further modification with ethanolamine in the synthesis of biobased polyurethane, thereby increasing wt % of biocontents in the final polymer. 1,4-Butanediol often used as a general chain extender in polyurethane synthesis was replaced partially with DV-EA. The generated polyurethane hard segment consists of DV-EA polyol and MDI (methylene diisocyanate) units or 1,4-butanediol and MDI units, respectively. The properties of the DV-EA-based polyurethane were investigated with differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic mechanical analyzer (DMA), X-ray diffraction spectroscopy (XRD), and universal testing machine (UTM). The results showed that this advanced polyurethane has 128% of Young’s modulus and 147% of increased strain compared to those of control, while its strength and thermal stability were maintained. It is expected that this new biobased tetraol may inspire a new perspective of vanillin application in biobased polyurethane synthesis