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

MOESM1 of In silico identification of metabolic engineering strategies for improved lipid production in Yarrowia lipolytica by genome-scale metabolic modeling

Additional file 1: Methods. MATLAB codes for revising metabolic model of Y. lipolytica Table S1. Comparison of eMOMA-predicted fluxes and 13C-MFA fluxes Table S2. List of candidate reactions for overexpression and knockout Table S3. Full list of predicted overexpression targets for increasing lipid production by more than 10% Table S4. List of strains and primers used in this study

Cryoprotective properties and preliminary characterization of exopolysaccharide (P-Arcpo 15) produced by the Arctic bacterium <i>Pseudoalteromonas elyakovii</i> Arcpo 15

Twenty-two bacterial strains that secrete exopolysaccharides (EPS) were isolated from marine samples obtained from the Chukchi Sea in the Arctic Ocean; of these, seven strains were found to be capable of producing cryoprotective EPS. The ArcPo 15 strain was isolated based on its ability to secrete large amounts of EPS, and was identified as Pseudoalteromonas elyakovii based on 16S rDNA analysis. The EPS, P-ArcPo 15, was purified by protease treatment and gel filtration chromatography. The purified EPS (P-ArcPo 15) had a molecular mass of 1.7 × 107 Da, and its infrared spectrum showed absorption bands of hydroxyl and carboxyl groups. The principal sugar components of P-ArcPo 15 were determined to be mannose and galacturonic acid, in the ratio of 3.3:1.0. The cryoprotective properties of P-ArcPo 15 were characterized by an Escherichia coli viability test. In the presence of 0.5% (w/v) EPS, the survival percentage of E. coli cells was as high as 94.19 ± 7.81% over five repeated freeze–thaw cycles. These biochemical characteristics suggest that the EPS P-ArcPo 15 may be useful in the development of cryoprotectants for biotechnological purposes, and we therefore assessed the utility of this novel cryoprotective EPS.</p

DataSheet1_Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine.docx

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5  h).</p

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

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>

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

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 1 of Generation of a recombinant antibody for sensitive detection of Pseudomonas aeruginosa

Additional file 1: Fig. S1. Complete original electrophoresis gel

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