Development of a graphene oxide nanosheet and double-stranded DNA structure based fluorescent "signal off" aptasensor for ochratoxin A detection in malt.

A "signal off" fluorescent aptasensor based on graphene oxide (GO) nanosheet and double-stranded DNA structure was fabricated for OTA detection. In the absence of OTA, the aptamer and its complementary DNA (cDNA) formed double-stranded conjugates that could coexist with GO, presenting fluorescence responses. Then, the presented OTA was captured by the aptamers, causing the release of cDNA-FAM probes. The free probes were adsorbed by GO, leading to an OTA concentration-dependent fluorescence quenching through fluorescence resonance energy transfer. Under optimum conditions, the fluorescent aptasensor exhibited outstanding sensitivity with a LOD of 11 pg/mL and a wide dynamic range of 0.04-30 ng/mL. The selectivity of the aptasensor was confirmed against other four mycotoxins, and the feasibility and reliability were verified by determining OTA in the spiked malt samples with satisfactory recovery of 95.50%-112.05%. This aptasensing platform could be adapted to measure other mycotoxins by replacing the aptamer sequences for food safety.

Recent progress in immunosensors for pesticides.

Immunosensors for rapid detection of pesticide residues has attracted considerable interest in the past few years for healthcare and environmental monitoring. And the publications have grown exponentially over the past decades, making it a trending hot-spot. Therefore, this review first examines the current situation regarding pesticide residue in various foods, feeds, traditional Chinese medicines and environmental samples. Then, the primary focus is on the recent development of the proposed immunosensors for pesticide detection in the past five years, with particular emphasis on the fluorescence, colorimetric, chemiluminescence, electrochemiluminescence, surface plasmon resonance, surface-enhanced Raman spectroscopy, electrochemical and piezoelectric sensors. Beyond a highlight of the real-world application and advantages of these emerging immunosensors for pesticide inspection, this paper also discusses their potential limits and current challenges, as well as future perspectives. This review will provide powerful insights to researchers for the future development of immunosensors, as well as their broader application in different fields, such as analytical chemistry, food safety control, clinical diagnostics, and environmental monitoring.

Developing a Novel Two-Stage Process for Carotenoid Production by Cordyceps militaris (Ascomycetes).

Natural carotenoids are attracting increasing interest, but their widespread use is limited because of poor production. Cordyceps militaris, a traditional Chinese mushroom, contains a large amount of carotenoids, and this study aimed to increase carotenoid production by C. militaris by optimizing a liquid-state cultivation system. We developed and optimized a novel 2-stage process, including cultivation under shaking in darkness and under static irradiation on a flat panel, using response surface methodology, and we compared this process to common shake-flask cultivation. In addition, we examined the effects of different inducers (chitosan, peanut oil, tomato juice, yeast, and metal ions) on carotenoid production. Results showed that under optimal conditions (4 days of shaking in darkness, 10 days of static irradiation with a 100-mL flat panel volume), a maximum of 1217.5 ± 115.9 µg/g carotenoids were produced; only 662.9 µg/g were produced by common shake-flask cultivation. Only a large amount of chitosan (8 mg) could significantly increase carotenoid content; some of the other inducers showed inhibitory effects. This study demonstrated that this 2-stage process could effectively increase the natural carotenoid content in C. militaris, making it a potential source for commercial exploitation.

Increasing of the Contain of Carotenoids in Caterpillar Mushroom, Cordyceps militaris (Ascomycetes) by Using the Fungal Elicitors Cultivation.

As a natural pigment, cordycepic carotenoids have many bioactive functions, such as antiinflammation, anticancer, and antioxidation. In addition, the good coloring of this hydrophilic pigment enables it to have wide application in the food industry. This study investigated five species of fungal elicitors, namely, Rhodotorula glutinis, Saccharomyces cerevisiae, Monascus ruber, Blakeslea trispora, and Flammulina velutipes, to evaluate their effects on carotenoid accumulation in Cordyceps militaris. Results showed that all fungal elicitors, except Rh. glutinis, have no positive effect on the biosynthesis of cordycepic carotenoids. The Rh. glutinis elicitor remarkably stimulated the accumulation of carotenoids with a 13.72% increase compared with the control. Subsequently, the entire Rh. glutinis elicitor (part NHK) was divided into three parts, namely, exopolysaccharide (EPS) (part E), mixture of EPS and protein (part PE), and other components (part O), to analyze their effects on carotenoid accumulations. Results showed that part O may be the effective component that remarkably stimulates the biosynthesis of carotenoids with a 26% increase compared with the control. This research demonstrated that Rh. glutinis elicitor can effectively increase the content of natural carotenoids in C. militaris, and provided an important reference for the development and utilization of carotenoid industrialization.

Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris.

Cordyceps militaris is a well-known edible medicinal mushroom in East Asia that contains abundant and diverse bioactive compounds. Since traditional genome editing systems in C. militaris were inefficient and complicated, here, we show that the codon-optimized cas9, which was used with the newly reported promoter Pcmlsm3 and terminator Tcmura3, was expressed. Furthermore, with the help of the negative selection marker ura3, a CRISPR-Cas9 system that included the Cas9 DNA endonuclease, RNA presynthesized in vitro and a single-strand DNA template efficiently generated site-specific deletion and insertion. This is the first report of a CRISPR-Cas9 system in C. militaris, and it could accelerate the genome reconstruction of C. militaris to meet the need for rapid development in the fungi industry.

Biosynthesis of Resveratrol in Blastospore of the Macrofungus Tremella fuciformis.

Tremella fuciformis is a known edible macrofungus that has medicinal value. It is widely cultivated in China and its products are distributed worldwide. In this study, a novel bioconversion system was established to produce resveratrol in the blastospore of T. fuciformis (bTf). The expression vector ptro1-4cl-rs that contains 4-coumarate:coenzyme A ligase gene (4cl) and resveratrol synthase gene (rs) was transformed into bTf by LiAc/PEG-mediated transformation. PCR and southern blotting analysis verified the successful integration of the exogenous 4cl and rs genes into the genome of bTf. HPLC analysis confirmed that two transformants can convert p-coumaric acid into resveratrol (0.92 and 0.83 µg/g resveratrol of dry weight within 7 days). This study is the first to report about the transformation and expression of resveratrol biosynthetic genes in bTf. This research is a significant step toward obtaining resveratrol-producing T. fuciformis strains, which not only satisfy the demand of resveratrol market, but also expand the category of functional food.

Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection.

Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4-coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p-coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans-resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermentation.

Compositional analysis of the fruiting body of transgenic Flammulina velutipes producing resveratrol.

Two strains of transgenic Flammulina velutipes TF71 and TF7H with 4cl and rs genes with the capability to produce resveratrol were obtained. In the nutrition assessment and analysis of transgenic strain and original strain (F7), the amino acid composition and proximate compositions of fruiting bodies were determined by amino acid automatic instrument and standard methods of the Association of Official Analytical Chemists (AOAC), while 4-coumaric acid, total flavonoids, and resveratrol were extracted by ethyl acetate and quantified by HPLC. Results indicated that significant differences were observed in proximate composition and amino acid between transgenic and original strains, but these detected components were within the normal ranges reported for F. velutipes. Total flavonoids and 4-coumaric acid contents of transgenic strains are lower than F7. Most important of all, resveratrol has been detected from TF71 and TF7H fruiting body but not found in F7, which was firstly produced by a transgenic mushroom.

Bioproduction of baccatin III, an advanced precursor of paclitaxol, with transgenic Flammulina velutipes expressing the 10-deacetylbaccatin III-10-O-acetyl transferase gene.

BACKGROUND: 10-Deacetylbaccatin III (10-DAB) and baccatin III are intermediates in the biosynthesis of Taxol (an anti-cancer drug) and useful precursors for semi-synthesis of the drug. In this study, a bioconversion system was established for the production of baccatin III, an advanced precursor of paclitaxel, in the transgenic mushroom Flammulina velutipes expressing the 10-deacetylbaccatin III-10ß-O-acetyltransferase gene. The expression vector pgFvs-TcDBAT containing the 10-deacetylbaccatin III-10ß-O-acetyltransferase (DBAT) gene was constructed and transformed into the cells of F. velutipes by polyethylene glycol-mediated protoplast transformation. RESULTS: Polymerase chain reaction and Southern blotting analysis verified the successful integration of the exogenous DBAT gene into the genome of F. velutipes. Reverse transcription polymerase chain reaction and enzyme activity analyses confirmed that the DBAT gene was expressed in F. velutipes, and DBAT is able to convert substrate into baccatin III. CONCLUSION: The DBAT gene from the plant Taxus chinensis can be functionally expressed in F. velutipes. Transgenic F. velutipes expressing the DBAT gene is able to produce the target product, baccatin III. This is the first report about the transformation and expression of paclitaxel biosynthetic gene in the edible mushroom F. velutipes. This represents a significant step towards bio-production of paclitaxel and its advanced precursor baccatin III in an edible fungus.

Composition of transgenic soybean seeds with higher γ-linolenic acid content is equivalent to that of conventional control.

γ-Linolenic acid (GLA) has been used as a general nutraceutical for pharmacologic applications, particularly in the treatment of skin conditions such as eczema. Four transgenic soybean lines that produce GLA at high yields (4.21% of total fatty acids, up to 1002-fold) were generated through the stable insertion of the Delta-6-fatty acid desaturase gene isolated from Borago officinalis into the genome of a conventional soybean cultivar. As part of the safety assessment of genetically engineered crops, the transgenic soybean seeds were compared with their parental soybean seeds (nontransgenic) by applying the principle of substantial equivalence. Compositional analyses were conducted by measuring the fatty acids, proximate analysis (moisture, crude protein, crude fat, carbohydrates, TDF, and ash contents), amino acids, lectins, and trypsin inhibitor activity. The present results showed that the specific transgenic cultivar studied was similar to the conventional control.