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.
It has been demonstrated that the results caused by abnormal levels of Cysteine (Cys) are not negligible in biological processes. Furthermore, mitochondria are one of the most important organelles, ...which not only produce cellular energy, but also participate in signal transmission, growth, death, and other biological processes. Therefore, developing an efficient fluorescent probe targeting mitochondria to detect Cys in cells is useful for clinic pathological diagnosis. In this report, a near-infrared (NIR) fluorescent probe LAA based on link-anthocyanin as fluorophore and acrylate as identification groups has been prepared. Link-anthocyanin, as an NIR-emitting fluorophore, showed favorable water solubility and strong fluorescence intensity. Acrylate, as a recognition site, exhibited intense sensitivity and selectivity to Cys. In addition, LAA had a high tendency to localize to mitochondria due to the lipophilic positive ion in the link-anthocyanin. Under the excitation of 560 nm, the probe LAA could detect Cys with high selectivity and low detection limit (37 nM) at the emission of 670 nm, which belonged to the NIR range and was beneficial for biological application. Furthermore, LAA exhibited great biocompatibility and low cytotoxicity, which could monitor Cys in mitochondria of living cells in real-time.
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.
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, Mβ, 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.
, 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
mycelium growth and conidia formation under nitrogen and phosphorus nutrient stress. The fatty acid profile of
was determined and the content of unsaturated fatty acid was found increased under nitrogen and phosphorus limitation. Oleic acid (C
), linoleic acid (C
), and γ-linolenic acid (C
) 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
. It also revealed that the molecular mechanisms of
respond to nitrogen and phosphorus stress. Our finding facilitates the construction of industrial strains with a nutrient-limited tolerance.
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 Al
3+
with turn-on fluorescent performance. As the concentration of Al
3+
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 Al
3+
was verified by Job's plot and MS analysis. The association constant between
1O
and Al
3+
was 3.9 × 10
2
mol
−1
L, and the limit of detection toward Al
3+
was 7.98 × 10
–9
mol L
−1
. Meanwhile, the probe can be utilized in practical water and logic circuits.
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
in
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
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
decreased the ergosterol content in
which may due to the feedback-mediated downregulation of
. Consistent with the decrease in ergosterol content, both
overexpression and RNAi strains of
are sensitive to abiotic stressors, including ergosterol biosynthesis inhibitor, temperature, salt and ethanol. Thus, we have identified the function of AoErg19 in
, which may assist in genetic modification of MVA and the ergosterol biosynthesis pathway.
Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of ...FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.
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.
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
. We investigated the effect of two oxidant agents (menadione sodium bisulfite, MSB, and hydrogen peroxide, H
O
) on cellular growth and antioxidant enzyme induction in
. 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.
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