Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection.

Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3'-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.

Transcriptome Study of Bursaphelenchus xylophilus Treated with Fomepizole Reveals a Serine/Threonine-Protein Phosphatase Gene that Is Substantially Linked with Vitality and Pathogenicity.

Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN.

Visual Detection of Dopamine with CdS/ZnS Quantum Dots Bearing by ZIF-8 and Nanofiber Membranes.

Dopamine (DA) is a widely present, calcium cholinergic neurotransmitter in the body, playing important roles in the central nervous system and cardiovascular system. Developing fast and sensitive DA detection methods is of great significance. Fluorescence-based methods have attracted much attention due to their advantages of easy operation, a fast response speed, and high sensitivity. This study prepared hydrophilic and high-performance CdS/ZnS quantum dots (QDs) for DA detection. The waterborne CdS/ZnS QDs were synthesized in one step using the amphiphilic polymer PEI-g-C14, obtained by grafting tetradecane (C14) to polyethyleneimine (PEI), as a template. The polyacrylonitrile nanofiber membrane (PAN-NFM) was prepared by electrospinning (e-spinning), and a metal organic frame (ZIF-8) was deposited in situ on the surface of the PAN-NFM. The CdS/ZnS QDs were loaded onto this substrate (ZIF-8@PAN-NFM). The results showed that after the deposition of ZIF-8, the water contact angle of the hydrophobic PAN-NFM decreased to within 40°. The nanofiber membrane loaded with QDs also exhibited significant changes in fluorescence in the presence of DA at different concentrations, which could be applied as a fast detection method of DA with high sensitivity. Meanwhile, the fluorescence on this PAN-NFM could be visually observed as it transitioned from a blue-green color to colorless, making it suitable for the real-time detection of DA.

Nematicidal Coumarins from Cnidium monnieri Fruits and Angelica dahurica Roots and Their Physiological Effect on Pine Wood Nematode (Bursaphelenchus xylophilus).

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is a major pathogen of pine wilt disease (PWD), which is a devastating disease affecting pine trees. Eco-friendly plant-derived nematicides against PWN have been considered as promising alternatives to control PWD. In this study, the ethyl acetate extracts of Cnidium monnieri fruits and Angelica dahurica roots were confirmed to have significant nematicidal activity against PWN. Through bioassay-guided fractionations, eight nematicidal coumarins against PWN were separately isolated from the ethyl acetate extracts of C. monnieri fruits and A. dahurica roots, and they were identified to be osthol (Compound 1), xanthotoxin (Compound 2), cindimine (Compound 3), isopimpinellin (Compound 4), marmesin (Compound 5), isoimperatorin (Compound 6), imperatorin (Compound 7), and bergapten (Compound 8) by mass and nuclear magnetic resonance (NMR) spectral data analysis. Coumarins 1-8 were all determined to have inhibitory effects on the egg hatching, feeding ability, and reproduction of PWN. Moreover, all eight nematicidal coumarins could inhibit the acetylcholinesterase (AChE) and Ca2+ ATPase of PWN. Cindimine 3 from C. monnieri fruits showed the strongest nematicidal activity against PWN, with an LC50 value of 64 µM at 72 h, and the highest inhibitory effect on PWN vitality. In addition, bioassays on PWN pathogenicity demonstrated that the eight nematicidal coumarins could effectively relieve the wilt symptoms of black pine seedlings infected by PWN. The research identified several potent botanical nematicidal coumarins for use against PWN, which could contribute to the development of greener nematicides for PWD control.

Transcriptome analysis of L-leucine-producing Corynebacterium glutamicum under the addition of trimethylglycine.

It has been widely reported that the addition of trimethylglycine (betaine) decreases osmotic pressure inhibition for cell growth, leading to increased production of amino acids. However, the underlying mechanism is unclear. To determine the global metabolic differences that occur under the addition of trimethylglycine, transcriptome analysis was performed. Transcriptome analysis of Corynebacterium glutamicum JL1211 revealed that 272 genes exhibited significant changes under trimethylglycine addition. We performed Gene Ontology (GO) and KEGG enrichment pathway analyses on these differentially expressed genes (DEGs). Significantly upregulated genes were mainly involved in the regulation of ABC transporters, especially phosphate transporters and sulfur metabolism. The three phosphate transporter genes pstC, pstA and pstB were upregulated by 13.06-fold, 29.80-fold and 30.49-fold, respectively. Notably, the transcriptional levels of the cysD, cysN, cysH and sir genes were upregulated by 81.5-fold, 57.3-fold, 77.6-fold and 125.4-fold, respectively, consistent with assimilatory sulfate reduction under the addition of trimethylglycine. The upregulation of ilvBN and leuD genes might result in increased L-leucine formation. The data indicated changes in the transcriptome of C. glutamicum with trimethylglycine treatment, thus providing a mechanism supporting the application of trimethylglycine in the production of L-leucine and other amino acids by C. glutamicum strains.

Dual-Aptamer-Assisted AND Logic Gate for Cyclic Enzymatic Signal Amplification Electrochemical Detection of Tumor-Derived Small Extracellular Vesicles.

Small extracellular vesicles (sEVs), often referred to as exosomes, are potential biomarkers for noninvasive cancer diagnosis. However, because of their phenotype heterogeneity, precise detection of tumor-derived sEVs is a great challenge. Herein, a dual-aptamer-assisted AND logic gate was fabricated for sensitive electrochemical detection of tumor-derived sEVs based on a cyclic enzymatic signal amplification strategy. Four different tumor-derived sEVs were used to verify the feasibility of the AND logic gate, and CCRF-CEM sEVs were successfully detected by this assay. The electrochemical assay shows a good linear response from 4 × 103 to 8 × 107 particles/µL, with a detection limit of 920 particles/µL, for CCRF-CEM sEVs, indicating potential application in accurate cancer diagnostics.

Electrochemical Sensing of Exosomal MicroRNA Based on Hybridization Chain Reaction Signal Amplification with Reduced False-Positive Signals.

MicroRNAs (miRNAs) in cancer cell-derived exosomes are important cancer biomarkers. Herein, a sensitive hybridization chain reaction (HCR) electrochemical assay was fabricated for the detection of exosomal microRNA-122 (miR-122). The hairpin DNA (hpDNA) probes were first immobilized on the surface of a gold electrode. In the presence of miR-122, the hairpin structure of the hpDNA could be opened and triggered the HCR through the cross-opening and hybridization of two helper DNA hairpins. Long nicked double helixes generated from HCR are used to capture more RuHex and increase the signal of differential pulse voltammetry (DPV). In this assay, the density of the hpDNA probes on the surface of the gold electrode was precisely controlled by the simultaneous immobilization of hpDNA and short 12 nucleotides single-stranded DNA (S-12), providing a very high amplification efficiency. More importantly, the false positive signal could be reduced or completely eliminated by applying exonuclease I (Exo I) before the introduction of target miR-122. Under optimal conditions, the assay offers very high sensitivity with an attomolar level detection limit, a linear range with 9 orders of magnitude, and specificity in single mismatch discrimination. This sensitive electrochemical assay could successfully evaluate the miR-122 concentration in different cancer-derived exosomes, indicating its potential use in cancer diagnostics.

A facile and label-free electrochemical aptasensor for tumour-derived extracellular vesicle detection based on the target-induced proximity hybridization of split aptamers.

Facile detection of tumour-derived extracellular vesicles (EVs) is crucial to cancer diagnosis. Herein, a facile and label-free electrochemical aptasensor was fabricated to detect tumour-derived EVs based on the target-induced proximity hybridization of split aptamers. In this assay, two designed oligonucleotide probes containing fragments of a protein tyrosine kinase-7 (PTK7) aptamer were used to recognize and capture EVs containing PTK7. In the presence of target EVs, the aptamer-target ternary complex could induce proximity hybridization and form a DNA duplex on the electrode. The DNA duplex could bind more electroactive Ru(NH3)63+ through electrostatic attraction, resulting in an increased cathodic current signal. By virtue of the excellent electrochemical signal reporter RuHex, the specificity of the aptamer and proximity ligation, a facile EV electrochemical aptasensor with a detection limit of 6.607 × 105 particles per mL was realized. Furthermore, this aptasensor showed good selectivity to distinguish different tumour-derived EVs and was applied to detect EVs in complex biological samples. The proposed electrochemical aptasensor can be further extended to the detection of other EVs, thus showing great potential in clinical diagnosis.

Transcriptomic analysis of Bursaphelenchus xylophilus treated by a potential phytonematicide, punicalagin.

Punicalagin showed significant nematotoxic activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, in the authors' previous research. The authors performed high-throughput transcriptomic sequencing of punicalagin-treated nematodes to generate clues for its nematotoxic mechanism of action. The authors identified 2,575 differentially expressed genes, 1,428 of which were up-regulated and 1,147 down-regulated. Based on a comprehensive functional in silico analysis, the authors speculate that PWN may respond to the stimulus of punicalagin through phagosome, endocytosis, peroxisome and MAPK signaling pathways. In addition, punicalagin could greatly affect PWN energy metabolism including oxidative phosphorylation. The genes encoding twitchin and a nematode cuticular collagen could be crucial regulation targets of punicalagin, which might contribute to its nematotoxic activity against PWN.Punicalagin showed significant nematotoxic activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, in the authors' previous research. The authors performed high-throughput transcriptomic sequencing of punicalagin-treated nematodes to generate clues for its nematotoxic mechanism of action. The authors identified 2,575 differentially expressed genes, 1,428 of which were up-regulated and 1,147 down-regulated. Based on a comprehensive functional in silico analysis, the authors speculate that PWN may respond to the stimulus of punicalagin through phagosome, endocytosis, peroxisome and MAPK signaling pathways. In addition, punicalagin could greatly affect PWN energy metabolism including oxidative phosphorylation. The genes encoding twitchin and a nematode cuticular collagen could be crucial regulation targets of punicalagin, which might contribute to its nematotoxic activity against PWN.

An electrochemiluminescent aptasensor for amplified detection of exosomes from breast tumor cells (MCF-7 cells) based on G-quadruplex/hemin DNAzymes.

Exosomes are non-invasive biomarkers for cancer diagnosis. Herein, we describe an electrochemiluminescent (ECL) aptasensor for the detection of exosomes from breast tumor cells. Mercaptopropionic acid (MPA)-modified Eu3+-doped CdS nanocrystals (MPA-CdS:Eu NCs) and H2O2 were used as ECL emitters and coreactant, respectively. The exosomes are recognized and captured by the CD63 aptamer, and then form a G-quadruplex/hemin DNAzyme, which efficiently catalyzes the decomposition of H2O2, resulting in the decreased ECL signal of MPA-CdS:Eu NCs. The exosomes from breast tumor cells (MCF-7 cells) can be detected in the range of 3.4 × 105 to 1.7 × 108 particles per mL. The limit of detection (LOD) was estimated to be 7.41 × 104 particles per mL at a signal-to-noise ratio of 3. The aptasensor has been successfully used to detect exosomes in the serum.

Dual-signal ratiometric electrochemiluminescence assay for detecting the activity of human methyltransferase.

In this paper, a dual-signal ratiometric electrochemiluminescence (ECL) assay was developed to detect human DNA (cytosine-5)-methyltransferase1 (DNMT1) activity. Mercaptopropionic acid (MPA)-modified Eu3+-doped CdS nanocrystals (MPA-CdS:Eu NCs) and luminol were used as two different ECL emitters, which showed ECL signals at different electrochemical potentials. The resultant ECL peaks exhibited opposite trends in the DNMT1 system. According to the ratio of the ECL responses of CdS to luminol, the DNMT1 activity was detected in the range of 1.0-30.0 U mL-1 in buffer solution. The limit of detection (LOD) was estimated to be 0.07 U mL-1 at a signal-to-noise ratio of 3. The assay was extended to detect the DNMT1 activity from crude lysates of cancer cells, along with the effect of DNMT1 inhibitors such as 5-aza and 5-aza-2'-dC.

Effects of betaine supplementation on L-threonine fed-batch fermentation by Escherichia coli.

Betaine can act as a stress protectant, methyl donor, or enzyme stabilizer in vitro for the biosynthesis of structurally complex compounds. The performances of betaine type and concentration on the metabolic processes of Escherichia coli JLTHR in a 5-L fermentor were investigated. The results showed that the maximum L-threonine production of 127.3 g/L and glucose conversion percentage of 58.12% was obtained fed with the glucose solution containing 2 g/L betaine hydrochloride, which increased by 14.5 and 6.87% more compared to that of the control, respectively. This study presents an analysis of the metabolic fluxes of E. coli JLTHR for the production of L-threonine with betaine supplementation. When betaine was fed into the fermentation culture medium, the metabolic flux entering into the pentose phosphate pathway (HMP) and biosynthesis route of L-threonine increased by 57.3 and 10.1%, respectively. In conclusion, exogenous addition of betaine was validated to be a feasible and efficacious approach to improve L-threonine production.

Nematotoxic coumarins from Angelica pubescens Maxim. f. biserrata Shan et Yuan roots and their physiological effects on Bursaphelenchus xylophilus.

The ethanol extracts from the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan was toxic against the pine wood nematode Bursaphelenchus xylophilus. The ethyl acetate-soluble fraction derived from this extract increased its potency with a mortality of 95.25% in 72 hr at 1.0 mg/mL. Four nematotoxic coumarins were obtained from the ethyl acetate extract by bioassay-guided isolation. These were identified as osthole 1, columbianadin 2, bergapten 3 and xanthotoxin 4 by mass and nuclear magnetic resonance spectral data analysis. The LC50 values against B. xylophilus in 72 hr were 489.17, 406.74, 430.08, and 435.66 µM, respectively. These compounds also altered the smooth morphology of the B. xylophilus exoskeleton to a rough and pitted appearance as visualized by electron microscopy. The coumarins 1-4 possessed significant acetylcholinesterase inhibitory activities but had negligible effects on amylase and cellulase. This research provides additional clues to the nematotoxic mechanism of coumarins against the pine wood nematode B. xylophilus. This work will assist in the development of coumarin nematicides with enhanced activity using molecular modifications of the core coumarin structure.The ethanol extracts from the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan was toxic against the pine wood nematode Bursaphelenchus xylophilus. The ethyl acetate-soluble fraction derived from this extract increased its potency with a mortality of 95.25% in 72 hr at 1.0 mg/mL. Four nematotoxic coumarins were obtained from the ethyl acetate extract by bioassay-guided isolation. These were identified as osthole 1, columbianadin 2, bergapten 3 and xanthotoxin 4 by mass and nuclear magnetic resonance spectral data analysis. The LC50 values against B. xylophilus in 72 hr were 489.17, 406.74, 430.08, and 435.66 µM, respectively. These compounds also altered the smooth morphology of the B. xylophilus exoskeleton to a rough and pitted appearance as visualized by electron microscopy. The coumarins 1-4 possessed significant acetylcholinesterase inhibitory activities but had negligible effects on amylase and cellulase. This research provides additional clues to the nematotoxic mechanism of coumarins against the pine wood nematode B. xylophilus. This work will assist in the development of coumarin nematicides with enhanced activity using molecular modifications of the core coumarin structure.

A nematicidal tannin from Punica granatum L. rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus).

The ethanol extract of Punica granatum L. rind was tested to show significant nematicidal activity against pine wood nematode. Three nematicidal compounds were obtained from the ethanol extract by bioassay-guided fractionation and identified as punicalagin 1, punicalin 2, and corilagin 3 by mass and nuclear magnetic resonance spectral data analysis. Punicalagin 1 was most active against PWN among the purified compounds with the LC50 value of 307.08µM in 72h. According to the enzyme assays in vitro, punicalagin 1 could inhibit the activity of acetylcholinesterase, amylase and cellulase from PWN with IC50 value of 0.60mM, 0.96mM and 1.24mM, respectively. The morphological structures of PWNs treated by punicalagin 1 were greatly changed. These physiological effects of punicalagin 1 on PWN may helpful to elucidate its nematicidal mechanism.

A thermostable xylanase hydrolyzes several polysaccharides from Bacillus altitudinis JYY-02 showing promise for industrial applications.

Polysaccharides have attracted immense attention as the largest source of bioactive compounds. Its bioavailability and bioactivity can be improved by utilizing degradation enzymes to reduce their molecular weight and viscosity. In this study, a 654 bp gene encoding xylanase was screened from the genome of Bacillus altitudinis JYY-02 and overexpressed in Escherichia coli Rosetta (DE3). The recombinant xylanase with a molecular weight of 27.98 kDa was purified (11.7-fold) using Ni-NTA affinity chromatography, with a 43.6% final yield. Through molecular docking, Glu, Arg, Tyr, and Trp were found to be the main amino acids involved in the interaction between xylanase and xylobiose. The effects of pH, temperature, metal ions, and substrates on xylanase activity were determined, and the results showed that the highest catalytic activity was displayed at pH 6.5, 50 °C temperature, with Cu2+ as an activator and xylan as the substrate. The Km (substrate concentration that yields a half-maximal velocity) and Vmax (maximum velocity) of recombinant xylanase were 6.876 mg/mL and 10984.183 µmol/mg∙pr/min, respectively. The recombinant xylanase was thermostable, with 85% and 39% of the enzymatic activity retained after 1 h at 60 °C and 1 h at 90 °C, respectively. The recombinant xylanase demonstrated a significant clarifying effect on fruit juices.

Fabrication and Characterization of Polylactic Acid Electrospun Wound Dressing Modified with Polyethylene Glycol, Rosmarinic Acid and Graphite Oxide.

Polylactic acid (PLA) is a biodegradable polymer made from natural sources, and its electrospinning (e-spinning) nanofiber membrane doped with antibacterial ingredients is widely used in the field of medical dressings. In this research, 9 wt% of rosmarinic acid (RosA) and 0.04 wt% of graphite oxide (GO) with synergistic antibacterial activity were introduced into the e-spinning PLA precursor solution, and the obtained PLA nanofiber membrane showed good antibacterial properties and wound healing effects. At the same time, a nonionic amphiphilic polymer, polyethylene glycol (PEG), was also introduced into this system to improve the hydrophilicity of the e-spinning membrane for wound healing application. The morphological characterization showed the RosA/GO and PEG did not affect the e-spinning of PLA. The tests of mechanical performance and wettability demonstrated that PEG and RosA/GO incorporated in PLA have migrated easily to the surface of the fiber. The e-spun PLA/PEG/RosA/GO membrane showed good antibacterial activity and promoted initial wound healing quickly, which would be a promising application in wound dressing.

Rosmarinic acid from eelgrass shows nematicidal and antibacterial activities against pine wood nematode and its carrying bacteria.

Pine wilt disease (PWD), a destructive disease for pine trees, is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus and additional bacteria. In this study, extracts of Zostera marina showed a high nematicidal activity against PWN and some of the bacteria that it carries. Light yellow crystals were obtained from extracts of Z. marina through solvent extraction, followed by chromatography on AB-8 resin and crystallization. The NMR and HPLC analysis showed that the isolated compound was rosmarinic acid (RosA). RosA showed effective nematicidal activity, of which the LC50 (50% lethal concentration) to PWN at 24 h, 48 h and 72 h was 1.18 mg/g, 1.05 mg/g and 0.95 mg/g, respectively. To get a high yield rate of RosA from Z. marina, single factor experiments and an L9 (34) orthogonal experiment were performed. This extraction process involved 70% ethanol for 3 h at 40 °C. The extraction dosage was 1:50 (w/v). The highest yield of RosA from Zostera was 3.13 mg/g DW (dried weight). The crude extracts of Zostera marina (10 mg/mL) and RosA (1 mg/mL) also showed inhibitory effects to some bacterial strains carried by PWN: Klebsiella sp., Stenotrophomonas maltophilia, Streptomyces sp. and Pantoea agglomerans. The results of these studies provide clues for preparing pesticide to control PWD from Z. marina.

[Isolation and characterization of Thermopirellula anaerolimosa gen. nov., sp. nov., an obligate anaerobic hydrogen-producing bacterium of the phylum Planctomycetes].

OBJECTIVE: To cultivate various yet-to-be cultured heterotrophs from anaerobic granule sludge, we used a selective culture medium with low concentrations of substrates supplemented a variety of antibiotics. METHODS: An obligate anaerobic, thermophilic, hydrogen-producing bacterium, strain VM20-7(T), was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating high-strength organic wastewater from isomerized sugar production processes. RESULTS: Cells of strain VM20-7(T) are non-motile, spherical, pear or teardrop shaped, occurring singly(o)r as aggregates (0.7 - 2.0 microm x 0.7 - 2.0 microm). Spore formation was not observed. Growth temperature ranges from 35 - 50 degrees C (optimum 45 degrees C), pH ranges from 6.0 - 8.3 (optimum 7.0 - 7.5) , NaCl tolerant concentration ranges from 0% - 0.5% (w/v, optimum 0% ). Nitrate, sulfate, thiosulfate, sulfite, elemental sulfur and Fe (III)-NTA were not used as terminal electron acceptors. Strain VM20-7(T) utilizes a wide range of carbohydrates, including glucose, maltose, ribose, xylose, sucrose, galactose, mannose, raffinose, pectin, yeast extract and xylan. Acetate and H2 are the main end products of glucose fermentation. The G + C content of the genomic DNA was 60.9 mol%. 16S rRNA gene sequence analysis revealed that it is related to the Pirellula-Rhodopirellula-Blastopirellula (PRB) clade within the order Planctomycetales (82.7 - 84.3% similarity with 16S rRNA genes of other known related species). CONCLUSION: The first obligate anaerobic bacterium within the phylum Planctomycetes was isolated with low concentration of carbohydrates and antibiotics. On the basis of the physiological and phylogenetic data, the name Thermopirellula anaerolimosa gen. nov. , sp. nov. is proposed for strain VM20-7(T) (= CGMCC 1.5169(T) = JCM 17478(T) = DSM 24165(T)).

Measurement of lactose concentration in milk by using engineered bacteria producing lycopene.

Lycopene is an orange-red carotenoid, which confers a visual phenotype to assess genetic transformation of microorganisms. In this study, the lycopene synthesis pathway was constructed in engineered Escherichia coli BL21 (DE3) by transforming plasmid pET-15b-crtBEI, wherein crtB, crtE, and crtI could be expressed under the control of the T7 promoter and lacO operator and lycopene could be accumulated in the engineered bacteria upon induction by lactose. A good linear relationship was observed between the lycopene content in engineered bacterial culture and lactose concentration in the range of 4-52 g/L; using this relation, the lactose concentration in milk could be determined. This method could be used to overcome several limitations of the high-performance liquid chromatography (HPLC) method for lactose detection, such as cumbersome sample preparation and expensive detection equipment. Moreover, this method required only a clean bench, shaker, and spectrophotometer for lactose analysis. Additionally, no significant difference was observed between this method and HPLC in terms of lactose measurement in milk, indicating that this method is reasonable and simple.

Isolation of the Thermostable ß-Glucosidase-Secreting Strain Bacillus altitudinis JYY-02 and Its Application in the Production of Gardenia Blue.

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by ß-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable ß-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of ß-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding ß-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant ß-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant ß-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-ß-d-glucopyranoside (pNPG) was used as the substrate. The recombinant ß-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE ß-Glucosidases are enzymes capable of hydrolyzing ß-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial ß-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable ß-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant ß-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified ß-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.