Near-infrared fluorescent probe for detection of hydrogen sulfide in water samples and food spoilage.

Hydrogen sulfide (H2S) is a common toxic gas that threatens the quality and safety of environmental water and food. Herein, a new near-infrared fluorescent probe DTCM was synthesized and characterized by single crystal X-ray diffraction for sensing H2S. It exhibited a remarkable "turn-on" near-infrared (NIR) emission response at 665 nm with a remarkably massive Stokes shift of 175 nm, super-rapid detection ability (within 30 s), excellent photostability, high selectivity and sensitivity (limit of detection, LOD = 58 nM). Additionally, the probe was successfully utilized for the detection of H2S in environmental water samples. The DTCM-loaded test papers enabled convenient and real-time monitoring of H2S produced by food spoilage.

Light regulation of secondary metabolism in fungi.

Fungi have evolved unique metabolic regulation mechanisms for adapting to the changing environments. One of the key features of fungal adaptation is the production of secondary metabolites (SMs), which are essential for survival and beneficial to the organism. Many of these SMs are produced in response to the environmental cues, such as light. In all fungal species studied, the Velvet complex transcription factor VeA is a central player of the light regulatory network. In addition to growth and development, the intensity and wavelength of light affects the formation of a broad range of secondary metabolites. Recent studies, mainly on species of the genus Aspergillus, revealed that the dimer of VeA-VelB and LaeA does not only regulate gene expression in response to light, but can also be involved in regulating production of SMs. Furthermore, the complexes have a wide regulatory effect on different types of secondary metabolites. In this review, we discussed the role of light in the regulation of fungal secondary metabolism. In addition, we reviewed the photoreceptors, transcription factors, and signaling pathways that are involved in light-dependent regulation of secondary metabolism. The effects of transcription factors on the production of secondary metabolites, as well as the potential applications of light regulation for the production of pharmaceuticals and other products were discussed. Finally, we provided an overview of the current research in this field and suggested potential areas for future research.

Molecular regulation of fungal secondary metabolism.

Many bioactive secondary metabolites synthesized by fungi have important applications in many fields, such as agriculture, food, medical and others. The biosynthesis of secondary metabolites is a complex process involving a variety of enzymes and transcription factors, which are regulated at different levels. In this review, we describe our current understanding on molecular regulation of fungal secondary metabolite biosynthesis, such as environmental signal regulation, transcriptional regulation and epigenetic regulation. The effects of transcription factors on the secondary metabolites produced by fungi were mainly introduced. It was also discussed that new secondary metabolites could be found in fungi and the production of secondary metabolites could be improved. We also highlight the importance of understanding the molecular regulation mechanisms to activate silent secondary metabolites and uncover their physiological and ecological functions. By comprehensively understanding the regulatory mechanisms involved in secondary metabolite biosynthesis, we can develop strategies to improve the production of these compounds and maximize their potential benefits.

Mitochondria-targeted fluorescent probe with long wavelength emission for detecting H2S and its application in foodstuff, water and living cells.

Hydrogen sulfide (H2S) is crucial to cellular energy production, apoptosis, and redox homeostasis in mitochondria of living cells. In this work, a unique mitochondria-targeting fluorescence probe (DDMI) was established for H2S determination based on styrylpyridinium scaffold. When DDMI was treated with H2S, it showed significant fluorescence enhancement at 623 nm, with good selectivity, and high sensitivity. In addition, the "turn-on" fluorescent probe DDMI could detect H2S in water samples with good recoveries in the range of 95.4 %-105.6 % and track the degree of food spoilage by visualizing the change of DDMI-loaded test strips. Furthermore, the established probe DDMI was successfully used for monitoring exogenous H2S in living cells and mitochondria targeting. These results paved the way for success in developing a technology that could be used to identify H2S in environment, foodstuff, and living cells.

A New High Selective and Sensitive Fluorescent Probe for Al3+ based on Photochromic Salicylaldehyde Hydrazyl Diarylethene.

A new diarylethene derivative 1O decorated with a salicylaldehyde hydrazine moiety was designed and synthesized successfully, and its structure was confirmed by NMR. Diarylethene 1O showed eminent photochromism and high selectivity and sensitivity for Al3+ with turn-on fluorescent performance. As the concentration of Al3+ in 1O solution increased, the color of solution remarkably changed from dark to bright green with 313-fold fluorescent emission intensity enhancement. The 1:1 combination stoichiometry between 1O and Al3+ was verified by Job's plot and MS analysis. The association constant between 1O and Al3+ was 3.9 × 102 mol-1 L, and the limit of detection toward Al3+ was 7.98 × 10-9 mol L-1. Meanwhile, the probe can be utilized in practical water and logic circuits.

A highly selective colorimetric and fluorescent probe Eu(tdl)2abp for H2S sensing: Application in live cell imaging and natural water.

Using 4-([2,2': 6', 2'- terpyridin] -4'-yl) -N, N-dimethylaniline (tdl) as auxiliary ligand and 6-azido-2,2'-bipyridine (abp) as recognition ligand, a europium complex fluorescent probe Eu(4-([2,2': 6', 2'-terpyridin] -4' -yl) -N, N-dimethylaniline)2-6-azido-2,2'-bipyridine Eu(tdl)2abp for efficient and specific recognition of hydrogen sulfide (H2S) was successfully synthesized and characterized by NMR and MS. Eu(tdl)2abp represented "on-off" fluorescence signals for H2S and its color changes could be identified with naked eyes. Eu(tdl)2abp had short response time (2 min) to H2S, high selectivity and good anti-interference, large stokes shift (207 nm). In various samples, when H2S existed, the azide group was reduced to amine group, resulting in closed fluorescence signal, and the fluorescence intensity reached the degree of quenching without being affected by other interference. At the same time, there was a good linear relationship between relative fluorescence intensity and H2S concentration with the detection limit (LOD) of 0.64 µM. The sensing mechanism of Eu(tdl)2abp to detect H2S was characterized by 1H NMR and HR-MS. Eu(tdl)2abp was used with success for the sensitive detection of H2S in natural water and living cells.

A coumarin-based fluorescent probe for specific detection of cysteine in the lysosome of living cells.

Cysteine (Cys), the only amino acid in the 20 natural amino acids that contains a reduced sulfhydryl group, plays important roles in the balance of redox homeostasis in biological systems. Lysosome is an important organelle containing a variety of hydrolases and has been proved to be the decomposition center of a variety of exogenous and endogenous macromolecular substances. In this research, a coumarin-based fluorescent probe MCA for the detection of Cys in lysosomes of living cells was developed. Due to the acrylate moiety, this probe exhibited high sensitivity (detection limit = 6.8 nM) and selectivity towards Cys superior to other analytes. Moreover, the probe was proved to be lysosome-targetable and showed good cell imaging ability and low cell toxicity.

Effects of Nitrogen and Phosphorus Limitation on Fatty Acid Contents in Aspergillus oryzae.

Aspergillus oryzae, commonly known as koji mold, has been widely used for the large-scale production of food products (sake, makgeolli, and soy sauce) and can accumulate a high level of lipids. In the present study, we showed the dynamic changes in A. oryzae mycelium growth and conidia formation under nitrogen and phosphorus nutrient stress. The fatty acid profile of A. oryzae was determined and the content of unsaturated fatty acid was found increased under nitrogen and phosphorus limitation. Oleic acid (C18:1), linoleic acid (C18:2), and γ-linolenic acid (C18:3) production were increased on five nitrogen and phosphorus limitation media, especially on nitrogen deep limitation and phosphorus limitation group, showing a 1. 2-, 1. 6-, and 2.4-fold increment, respectively, compared with the control. Transcriptomic analysis showed the expression profile of genes related to nitrogen metabolism, citrate cycle, and linoleic acid synthesis, resulting in the accumulation of unsaturated fatty acid. qRT-PCR results further confirmed the reliability and availability of the differentially expressed genes obtained from the transcriptome analysis. Our study provides a global transcriptome characterization of the nitrogen and phosphorus nutrient stress adaptation process in A. oryzae. It also revealed that the molecular mechanisms of A. oryzae respond to nitrogen and phosphorus stress. Our finding facilitates the construction of industrial strains with a nutrient-limited tolerance.

Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi.

Filamentous fungi possess the capacity to produce a wide array of secondary metabolites with diverse biological activities and structures, such as lovastatin and swainsonine. With the advent of the post-genomic era, increasing amounts of cryptic or uncharacterized secondary metabolite biosynthetic gene clusters are continually being discovered. However, owing to the longstanding lack of versatile, comparatively simple, and highly efficient genetic manipulation techniques, the broader exploration of industrially important secondary metabolites has been hampered thus far. With the emergence of CRISPR/Cas9-based genome editing technology, this dilemma may be alleviated, as this advanced technique has revolutionized genetic research and enabled the exploitation and discovery of new bioactive compounds from filamentous fungi. In this review, we introduce the CRISPR/Cas9 system in detail and summarize the latest applications of CRISPR/Cas9-mediated genome editing in filamentous fungi. We also briefly introduce the specific applications of the CRISPR/Cas9 system and CRISPRa in the improvement of secondary metabolite contents and discovery of novel biologically active compounds in filamentous fungi, with specific examples noted. Additionally, we highlight and discuss some of the challenges and deficiencies of using the CRISPR/Cas9-based genome editing technology in research on the biosynthesis of secondary metabolites as well as future application of CRISPR/Cas9 strategy in filamentous fungi are highlighted and discussed.

A Turn-on Fluorescent Probe for the Discrimination of Cys/Hcy and GSH With Dual Emission Signals.

In this paper, we successfully synthesized a simple and versatile fluorescent probe. This probe was not only easily prepared with a high yield, but also showed rapid selective and sensitive responses for Cys/Hcy and GSH. The probe can be used as a naked-eye detector for Cys/Hcy and GSH from other analytes. As a fluorescent sensor, it can be used to simultaneously detect and discriminate Cys/Hcy from GSH with two fluorescent emission signals without spectral crosstalk.

A novel full symmetric diarylethene-based ratiometric fluorescent sensor for lysine and the application for a logic circuit.

A novel diarylethene-based ratiometric fluorescent sensor with full symmetric structure, 1o, was designed and synthesized successfully. 1o could identify lysine (Lys) with high selectivity and sensitivity and the fluorescence emission peak was red shifted 85 nm upon addition of Lys, which could realize ratio recognition. It exhibited excellent anti-interference performance in the presence of various amino acids in CH3 CN/H2 O (7/3, v/v) solution. Moreover, the limit of detection of 1o to Lys could reach 0.019 µM based on a good linear range of 0-40 µM. In addition, the fluorescence emission intensity of 1o could be turned off/on by ultraviolet/visible light due to the special structure of diarylethene. A logic circuit was designed with three inputs. The ratiometric fluorescent sensor 1o could be as a new tool and provide a new method for detection of Lys.

Identification and Expression Analysis of Sugar Transporter Gene Family in Aspergillus oryzae.

Sugar transporter (SUT) genes are associated with multiple physiological and biochemical processes in filamentous fungi, such as the response to various stresses. However, limited systematic analysis and functional information of SUT gene family have been available on Aspergillus oryzae (A. oryzae). To investigate the potential roles of SUTs in A. oryzae, we performed an integrative analysis of the SUT gene family in this study. Based on the conserved protein domain search, 127 putative SUT genes were identified in A. oryzae and further categorized into eight distinct subfamilies. The result of gene structure and conserved motif analysis illustrated functional similarities among the AoSUT proteins within the same subfamily. Additionally, expression profiles of the AoSUT genes at different growth stages elucidated that most of AoSUT genes have high expression levels at the stationary phase while low in the adaptive phase. Furthermore, expression profiles of AoSUT genes under salt stress showed that AoSUT genes may be closely linked to salt tolerance and involved in sophisticated transcriptional process. The protein-protein interaction network of AoSUT propounded some potentially interacting proteins. A comprehensive overview of the AoSUT gene family will offer new insights into the structural and functional features as well as facilitate further research on the roles of AoSUT genes in response to abiotic stresses.

Genome-wide identification and expression profile of the MADS-box gene family in Erigeron breviscapus.

The MADS-box gene family encodes transcription factors with many biological functions that extensively regulate plant growth, development and reproduction. Erigeron breviscapus is a medicinal herb used widely in traditional Chinese medicine, and is believed to improve blood circulation and ameliorate platelet coagulation. In order to gain a detailed understanding of how transcription factor expression may regulate the growth of this potentially important medicinal plant, a genome-wide analysis of the MADS-box gene family of E. breviscapus is needed. In the present study, 44 MADS-box genes were identified in E. breviscapus and categorized into five subgroups (MIKC, Mα, Mß, Mγ and Mδ) according to their phylogenetic relationships with the Arabidopsis MADS-box genes. Additionally, the functional domain, subcellular location and motif compositions of the E. breviscapus MADS-box gene products were characterized. The expression levels for each of the E. breviscapus MADS-box (EbMADS) genes were analyzed in flower, leaf, stem and root organs, and showed that the majority of EbMADS genes were expressed in flowers. Meanwhile, some MADS genes were found to express high levels in leaf, stem and root, indicating that the MADS-box genes are involved in various aspects of the physiological and developmental processes of the E. breviscapus. The results from gene expression analysis under different pollination treatments revealed that the MADS-box genes were highly expressed after non-pollinated treatment. To the best of our knowledge, this study describes the first genome-wide analysis of the E. breviscapus MADS-box gene family, and the results provide valuable information for understanding of the classification, cloning and putative functions of the MADS-box family.

Oxidative Stress Response of Aspergillus oryzae Induced by Hydrogen Peroxide and Menadione Sodium Bisulfite.

Oxidative stress response protects organisms from deleterious effects of reactive oxygen species (ROS), which can damage cellular components and cause disturbance of the cellular homeostasis. Although the defensive biochemical mechanisms have been extensively studied in yeast and other filamentous fungi, little information is available about Aspergillus oryzae. We investigated the effect of two oxidant agents (menadione sodium bisulfite, MSB, and hydrogen peroxide, H2O2) on cellular growth and antioxidant enzyme induction in A. oryzae. Results indicated severe inhibition of biomass and conidia production when high concentration of oxidants was used. Transcriptomic analysis showed an up-regulated expression of genes involved in oxidoreduction, such as catalase, glutathione peroxidase, and superoxide dismutase. In addition, it was observed that oxidative stress stimuli enhanced the expression of Yap1 and Skn7 transcription factors. Further, metabolomic analysis showed that glutathione content was increased in the oxidative treatments when compared with the control. Moreover, the content of unsaturated fatty acid decreased with oxidative treatment accompanying with the down-regulated expression of genes involved in linoleic acid biosynthesis. This study provided a global transcriptome characterization of oxidative stress response in A. oryzae, and can offer multiple target genes for oxidative tolerance improvement via genetic engineering.

Hg2+ -selective ratiometric and colorimetric probe based on dansyl-rhodamine and its staining function in cell imaging.

A new ratiometric probe composed of a dansyl-rhodamine dyad for the detection of Hg2+ via fluorescence resonance energy transfer was designed and synthesized. Rhodamine, dansyl chloride, and hydrazide were selected as the acceptor, donor, and reaction site, respectively. It displayed high selectivity and sensitivity to Hg2+ with obvious colour change and fluorescence change due to Hg2+ -assisted hydrolysis of rhodamine hydrazide. A good linear relationship ranging from 0 to 16 µM and 0-28 µM for the Hg2+ concentration was found based on absorbance and fluorescence assay, respectively. Detection limits of absorbance and fluorescence for Hg2+ were calculated to be 1.22 µM and 9.10 µM, respectively.

Mevalonate Diphosphate Decarboxylase MVD/Erg19 Is Required for Ergosterol Biosynthesis, Growth, Sporulation and Stress Tolerance in Aspergillus oryzae.

Mevalonate diphosphate decarboxylase (MVD; EC 4.1.1.33) is a key enzyme of the mevalonic acid (MVA) pathway. In fungi, the MVA pathway functions as upstream of ergosterol biosynthesis, and MVD is also known as Erg19. Previously, we have identified Aoerg19 in Aspergillus oryzae using bioinformatic analysis. In this study, we showed that AoErg19 function is conserved using phylogenetic analysis and yeast complementation assay. Quantitative reverse transcription-PCR (qRT-PCR) indicated that Aoerg19 expression changed in different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg19 was located in the vacuole. Overexpression of Aoerg19 decreased the ergosterol content in A. oryzae, which may due to the feedback-mediated downregulation of Aoerg8. Consistent with the decrease in ergosterol content, both Aoerg19 overexpression and RNAi strains of A. oryzae are sensitive to abiotic stressors, including ergosterol biosynthesis inhibitor, temperature, salt and ethanol. Thus, we have identified the function of AoErg19 in A. oryzae, which may assist in genetic modification of MVA and the ergosterol biosynthesis pathway.

Functional Genomics of Aspergillus oryzae: Strategies and Progress.

Aspergillus oryzae has been used for the production of traditional fermentation and has promising potential to produce primary and secondary metabolites. Due to the tough cell walls and high drug resistance of A. oryzae, functional genomic characterization studies are relatively limited. The exploitation of selection markers and genetic transformation methods are critical for improving A. oryzae fermentative strains. In this review, we describe the genome sequencing of various A. oryzae strains. Recently developed selection markers and transformation strategies are also described in detail, and the advantages and disadvantages of transformation methods are presented. Lastly, we introduce the recent progress on highlighted topics in A. oryzae functional genomics including conidiation, protein secretion and expression, and secondary metabolites, which will be beneficial for improving the application of A. oryzae to industrial production.

A colorimetric and fluorescent chemosensor based on diarylethene for simultaneous detection and discrimination of biothiols.

In this work, a novel probe D-HBT-NBD (1O) based on diarylethene to detect biothiols (including Cys, Hcy and GSH) was synthesized and the relative colorimetric and fluorescent properties were tested. The probe exhibited excellent photochromic properties and showed apparent colorimetric and fluorescent signals for Cys, Hcy and GSH. The probe can selectively detect Cys, Hcy and GSH by naked eyes for its open-ring isomer 1O and can discriminate Cys from Hcy/GSH by apparent color change from light orange to dark pink at the closed-ring state under the irradiation of UV light. At the excitation wavelength of 465 nm, the probe could be used to discriminate GSH from Cys/Hcy with no fluorescent emission at 570 nm. Taking advantage of the photochromic property of the diarylethene moiety and the different fluorescent properties of NBD derivatives of GSH and Cys/Hcy, 1O could be used as a novel probe to discriminate Cys, Hcy and GSH from each other simultaneously. Meanwhile, a logic gate was constructed based on the colorimetric and fluorescent properties of 1O.

Heterologous expression of AoD9D enhances salt tolerance with increased accumulation of unsaturated fatty acid in transgenic Saccharomyces cerevisiae.

Salt stress can trigger several physiological responses in microorganisms such as the increasing accumulation of unsaturated fatty acid, which was biosynthesized by delta-9 fatty acid desaturases (D9D) at the first step. In the present study, two D9D genes, designated AoD9D1 and AoD9D2, were isolated from Aspergillus oryzae. The expression analysis showed that AoD9D1 and AoD9D2 were upregulated under salt stress. To investigate the function of AoD9D, transgenic Saccharomyces cerevisiae strains that heterologously expressed AoD9D were exposed to salinity condition. These transgenic strains exhibited greater tolerance to salt stress than wild-type strains, and the heterologous expression of AoD9D increased the content in unsaturated fatty acids as compared to control cells. Moreover, AoD9D1 and AoD9D2 both contained fatty acid desaturase (FAD) and cytochrome b5-like Heme/Steroid-binding domains (Cyt-b5). S. cerevisiae separately transformed with the gene fragments coding for the FAD and Cyt-b5 domains in the AoD9D1 protein grew better and accumulated a higher concentration of unsaturated FAs than the control. Altogether, the heterologous expression of AoD9D enhanced the tolerance of transgenic S. cerevisiae to high salinity stress with increased accumulation of unsaturated fatty acid. The results provide some practical basis for the successful development of salt-tolerant fermentation microorganisms.

Aggregation-induced emission enhancement (AIEE)-active tetraphenylethene (TPE)-based chemosensor for Hg2+ with solvatochromism and cell imaging characteristics.

An aggregation-induced emission enhancement (AIEE)-active fluorescent sensor based on a tetraphenylethene (TPE) unit has been successfully designed and synthesized. Interestingly, the luminogen could detect Hg2+ with high selectivity in an acetonitrile solution without interference from other competitive metal ions, and the detection limit was 7.46 × 10-6 mol L-1. Furthermore, the luminogen also showed interesting solvatochromic behavior and superior cell imaging performance.