The serine/threonine protein kinase MpSTE1 directly governs hyphal branching in Monascus spp.

Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)-assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites. KEY POINTS: • MpSTE1 is the first characterized regulator for tightly regulating hyphal branching • Overexpression of mpSTE1 significantly improves secondary metabolite production • A high-throughput image analysis tool was developed for counting hyphal branching.

Clinical and molecular epidemiologic features of enterovirus D68 infection in children with acute lower respiratory tract infection in China.

Acute flaccid paralysis (AFP) associated with enterovirus D68 (EV-D68) infection has attracted much attention since an outbreak in the USA in 2014. Notably, EV-D68 was detected in a child with AFP for the first time in China in 2018. In a multicentre study from May 2017 to December 2019, we monitored EV-D68 infections in hospitalized children with acute lower respiratory tract infection (ALRTI) in China. Out of 3,071 samples collected from patients with ALRTI, ten were positive for EV-D68. All patients presented with mild diseases with no neurological symptoms or signs. Phylogenetic analysis based on the VP1 gene showed that all EV-D68 sequences obtained in this study belonged to subclade B3 and were close to sequences of EV-D68 strains obtained from patients with AFP in the USA. Four EV-D68 strains were isolated, and their complete genome sequences were determined. These sequences did not show any evidence of recombination events. To assess their neurotropism, the isolates were used to infect the "neuronal-like" cell line SH-SY5Y, and resulted in a cytopathic effect. We further analysed the structure and sites that may be associated with neurovirulence, including the stem-loop structure in the untranslated region (3'UTR) and identified amino acid substitutions (M291T, V341A, T860N, D927N, S1108G, and R2005K) in the coding region and specific nucleotides (127T, 262C, and 339T) in the 5' UTR. In conclusion, EV-D68 infection was detected in a small number of children with ALRTI in China from 2017 to 2019. Disease symptoms in these children were relatively mild with no neurological complications, and all EV-D68 sequences belonged to subclade B3.

An Aerolysin-like Pore-Forming Protein Complex Targets Viral Envelope to Inactivate Herpes Simplex Virus Type 1.

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.

A novel N-arachidonoyl-l-alanine-catabolizing strain of Serratia marcescens for the bioremediation of Cd and Cr co-contamination.

Cadmium (Cd) and chromium (Cr) are widespread contaminants with a high risk to the environment and humans. Herein we isolated a novel strain of Serratia marcescens, namely strain S27, from soil co-contaminated with Cd and Cr. This strain showed strong resistance to Cd as well as Cr. S27 cells demonstrated Cd adsorption rate of 45.8% and Cr reduction capacity of 84.4% under optimal growth conditions (i.e., 30 °C, 200 rpm, and pH 7.5). Microscopic characterization of S27 cells revealed the importance of the functional groups C-O-C, C-H-O, C-C, C-H, and -OH, and also indicated that Cr reduction occurred on bacterial cell membrane. Cd(II) and Cr(VI) bioaccumulation on S27 cell surface was mainly in the form of Cd(OH)2 and Cr2O3, respectively. Further, metabolomic analyses revealed that N-arachidonoyl-l-alanine was the key metabolite that promoted Cd and Cr complexation by S27; it primarily promotes γ-linolenic acid (GLA) metabolism, producing siderophores and coordinating with organic acids to enhance metal bioavailability. To summarize, our results suggest that S27 is promising for the bioremediation of environments contaminated with Cd and Cr in tropical regions.

Dynamic regulation of Monascus azaphilones biosynthesis by the binary MrPigE-MrPigF oxidoreductase system.

Monascus azaphilones (MAs) have been extensively applied as natural food coloring agents. MAs are classified into three categories: yellow MAs (YMAs), orange MAs (OMAs), and red MAs with various biological activities. However, the exact biosynthetic mechanism of OMAs and YMAs are not thoroughly elucidated. Firstly, we identified four DNA-binding residues of transcription factor MrPigB and constructed a multi-site saturation mutagenesis library of MrPigB. Then, comparative metabolite and gene expression of the mutants revealed that two oxidoreductases MrPigE and MrPigF were responsible for the formation of YMAs and OMAs. Finally, the in vitro and in vivo assays demonstrated the opposite roles of MrPigE and MrPigF in conversion of OMAs to YMAs. To our knowledge, this is the first report of a binary oxidoreductase system for dynamic regulation of fungal secondary metabolite biosynthesis. Broadly, our work also demonstrates the transcription factor engineering strategy for elucidating the biosynthetic pathway of secondary metabolite. KEY POINTS: • MrPigE converts orange Monascus azaphilones to yellow Monascus azaphilones • MrPigF oxidizes intermediates to afford orange Monascus azaphilones • MrPigE and MrPigF constitute a binary system in Monascus azaphilones biosynthesis.

Comparison of clinical features of acute lower respiratory tract infections in infants with RSV/HRV infection, and incidences of subsequent wheezing or asthma in childhood.

BACKGROUND: To compare the clinical characteristics of acute lower respiratory tract infections (ALRTIs) caused by respiratory syncytial virus (RSV) and human rhinovirus (HRV) and to explore the relationship between the development of recurrent wheezing/asthma and RSV/ HRV infections in infancy. METHODS: Retrospective study was conducted to compare the clinical characteristics of acute lower respiratory tract infections (ALRTIs). Hospitalized patients with ALRTIs from March 2007 to December 2016 were screened. Single RSV cases (s-RSV), single HRV cases (s-HRV), and cases who had co-infection with the two viruses were enrolled. Follow-up was performed to determine whether either specific respiratory virus infection was related to subsequent development of recurrent wheezing/asthma. RESULTS: The s-RSV children were the youngest (P = 0.021), they experienced the most serious condition (P < 0.001) and respiratory failure (P < 0.001), they also required highest demand of oxygen therapy (P < 0.001). And in s-RSV group, the incidence of development of recurrent wheezing was significantly higher in subgroup with the family history of wheezing than that without (P < 0.001). CONCLUSION: The s-RSV cases suffered from the worst severity of illness, respiratory failure and required highest demand of oxygen therapy. Recurrent wheezing was more common in s-RSV group with family history of wheezing than those without.

Association of multiple candidate genes with mild cognitive impairment in an elderly Chinese Uygur population in Xinjiang.

BACKGROUND: Mild cognitive impairment (MCI) is a high-risk factor for Alzheimer's disease (AD). In the present study, we investigated the association of genetic polymorphisms of five genes (8-oxoguanine DNA glycosylase 1 (OGG1), bridging integrator 1 (BIN1), sortilin-related receptor 1 (SORL1), presenilin 2 (PSEN2) and nerve growth factor (NGF)) with MCI risk in a Xinjiang Uygur population. We also tested the relationship between the promoter methylation of genes OGG1 and dihydrolipoamide S-succinyltransferase (DLST) with MCI. METHODS: This study involved 43 MCI patients and 125 controls. Genotyping was done by Sanger sequencing. DNA methylation assays used quantitative methylation-specific polymerase chain reaction. RESULTS: We found that polymorphisms of five genes and the methylation of DLST and OGG1 genes were not associated with MCI (P > 0.05). Further subgroup analysis found that DLST hypomethylation was significantly associated with MCI in the carriers of apolipoprotein E (APOE) ε4 (P = 0.042). In the carriers of non-APOE ε4, DLST methylation levels were significantly lower in the male control group than in the female control group (p = 0.04). Meanwhile, among the non-APOE ε4 carriers younger than 75, OGG1 hypermethylation levels were significantly associated with MCI (P = 0.049). DLST methylation in female controls was significantly lower than that in male controls (P = 0.003). According to gender stratification, there was a significant positive correlation of fasting plasma glucose (FBG) and high-density lipoprotein (HDL) with OGG1 methylation in the female controls (FBG: P = 0.024; HDL: P = 0.033). There was a significant inverse correlation between low-density lipoprotein and DLST methylation in male MCI (P = 0.033). There was a significant positive correlation between HDL and DLST methylation levels in the female controls (P = 0.000). CONCLUSIONS: This study was the first to discover that DLST promoter methylation interacted with APOE ε4 and thus affected the pathogenesis of MCI. In addition, OGG1 promoter methylation interacted with several other factors to increase the risk of MCI.

Case-control study of the epidemiological and clinical features of human adenovirus 55 and human adenovirus 7 infection in children with acute lower respiratory tract infections in Beijing, China, 2008-2013.

BACKGROUND: In adults, the emerging human adenovirus (HAdV) type 55 (HAdV-55) has been reported to cause more severe cases of adenovirus induced acute lower respiratory tract infections (ALRTIs) compared to other HAdV serotypes (HAdV-3, HAdV-7, HAdV-14). However, there is a dearth of comparative studies in children that address differences in the clinical epidemiological features between HAdV-55 and other HAdV serotypes that can also induce severe infection (such as HAdV-7). METHODS: We conducted a retrospective review of pediatric patients hospitalized at Beijing Children's Hospital with ALRTI from April 2008 to December 2013 who had adenovirus detected from nasopharyngeal or throat samples by PCR. We further compared pediatric patients infected with HAdV-55 to those infected with HAdV-7 using a case-control methodology by matching each subject with HAdV-55 infection to 4 patients with HAdV-7 infection within 2 months of each HAdV-55 infection. Demographic, clinical, and etiological data were collected and analyzed. RESULTS: Over the five-year period, HAdV was detected in 194 children. Of these, 8 were HAdV-55 positive. Epidemiological results showed that HAdV-55 infection was observed only in 4% of adenovirus infected children whereas HAdV-7 infection proportioned 53%. Most cases of HAdV-55 infection were identified during March and April, whereas HAdV-7 infection occurred throughout the year. Wheezing was significantly less frequent in the HAdV-55 group. No patients infected with HAdV-55 presented with vomiting or had any underlying disease. Coinfections with other respiratory tract pathogens were frequent among children infected with either HAdV-55 or HAdV-7. CONCLUSIONS: HAdV-55 circulated in Beijing during spring and appeared to cause pediatric respiratory infections that were as severe as HAdV-7 infections. Broader surveillance studies are needed.

A turn-on fluorescent aptasensor for adenosine detection based on split aptamers and graphene oxide.

A simple, sensitive and selective turn-on fluorescent aptasensor for adenosine detection was developed based on target-induced split aptamer fragment conjunction and different interactions of graphene oxide and the two states of the designed aptamer sequences.

Monaspin B, a Novel Cyclohexyl-furan from Cocultivation of Monascus purpureus and Aspergillus oryzae, Exhibits Potent Antileukemic Activity.

Natural products are a rich resource for the discovery of innovative drugs. Microbial cocultivation enables discovery of novel natural products through tandem enzymatic catalysis between different fungi. In this study, Monascus purpureus, as a food fermentation strain capable of producing abundant natural products, was chosen as an example of a cocultivation pair strain. Cocultivation screening revealed that M. purpureus and Aspergillus oryzae led to the production of two novel cyclohexyl-furans, Monaspins A and B. Optimization of the cocultivation mode and media enhanced the production of Monaspins A and B to 1.2 and 0.8 mg/L, respectively. Monaspins A and B were structurally elucidated by HR-ESI-MS and NMR. Furthermore, Monaspin B displayed potent antiproliferative activity against the leukemic HL-60 cell line by inducing apoptosis, with a half-maximal inhibitory concentration (IC50) of 160 nM. Moreover, in a mouse leukemia model, Monaspin B exhibited a promising in vivo antileukemic effect by reducing white blood cell, lymphocyte, and neutrophil counts. Collectively, these results indicate that Monaspin B is a promising candidate agent for leukemia therapy.

Trends in phytoremediation of heavy metals-contaminated soils: A Web of science and CiteSpace bibliometric analysis.

Heavy metals pollution in soils is an urgent environmental issue worldwide. Phytoremediation is a green and eco-friendly way of remediating heavy metals. However, a systematic overview of this field is limited, and little is known about future development trends. Therefore, we used CiteSpace software to conduct bibliometric and visual analyses of published literature in the field of phytoremediation of heavy metals in soils from the Web of Science core collection and identified research hotspots and development trends in this field. Researchers are paying increased attention to phytoremediation of heavy metals in soils, especially environmental researchers. A total of 121 countries or regions, 3790 institutions, 4091 funded organisations and 15,482 authors have participated in research in this area. China, India, and Pakistan are the largest contributors. There has been extensive cooperation between countries, institutions, and authors worldwide, but there is a lack of cooperation among top authors. 'Calcareous soil', 'Co-contaminated soil' and 'Metal availability' are the most intensively investigated topics. 'EDTA', 'Plant growth-promoting Rhizobacteria', 'Photosynthesis', 'Biochar' and 'Phytoextraction' are research hotspots in this field. In addition, more and more researchers are beginning to pay attention to research on co-contaminated soil, metal availability, chelating agents, and microbial-assisted phytoremediation. In summary, bibliometric, and visual analyses in the field of phytoremediation of heavy metals in soils identifies probable directions for future research and provides a resource through which to better understand this rapidly advancing subject.

Activation of the NLRP3 inflammasome by human adenovirus type 7 L4 100-kilodalton protein.

Human adenovirus type 7 (HAdV-7) is a significant viral pathogen that causes respiratory infections in children. Currently, there are no specific antiviral drugs or vaccines for children targeting HAdV-7, and the mechanisms of its pathogenesis remain unclear. The NLRP3 inflammasome-driven inflammatory cascade plays a crucial role in the host's antiviral immunity. Our previous study demonstrated that HAdV-7 infection activates the NLRP3 inflammasome. Building upon this finding, our current study has identified the L4 100 kDa protein encoded by HAdV-7 as the primary viral component responsible for NLRP3 inflammasome activation. By utilizing techniques such as co-immunoprecipitation, we have confirmed that the 100 kDa protein interacts with the NLRP3 protein and facilitates the assembly of the NLRP3 inflammasome by binding specifically to the NACHT and LRR domains of NLRP3. These insights offer a deeper understanding of HAdV-7 pathogenesis and contribute to the development of novel antiviral therapies.

Effect of EDDS on the rhizosphere ecology and microbial regulation of the Cd-Cr contaminated soil remediation using king grass combined with Piriformospora indica.

The negative impacts of soil heavy metals composite pollution on agricultural production and human health are becoming increasingly prevalent. The applications of green chelating agents and microorganisms have emerged as promising alternate methods for enhancing phytoremediation. The regulatory effects of root secretion composition, microbial carbon source utilization, key gene expression, and soil microbial community structure were comprehensively analyzed through a combination of HPLC, Biolog EcoPlates, qPCR, and high-throughput screening techniques. The application of EDDS resulted in a favorable rhizosphere ecological environment for the king grass Piriformospora indica, characterized by a decrease in soil pH by 0.41 units, stimulation of succinic acid and fumaric acid secretion, and an increase in carbon source metabolic activity of amino acids and carbohydrates. Consequently, this improvement enhanced the bioavailability of Cd/Cr and increased the biomass of king grass by 25.7%. The expression of dissimilatory iron-reducing bacteria was significantly upregulated by 99.2%, while there was no significant difference in Clostridium abundance. Furthermore, the richness of the soil rhizosphere fungal community (Ascomycota: 45.8%, Rozellomycota: 16.7%) significantly increased to regulate the proportion of tolerant microbial dominant groups, promoting the improvement of Cd/Cr removal efficiency (Cd: 23.4%, Cr: 18.7%). These findings provide a theoretical basis for the sustainable development of chelating agent-assisted plants-microorganisms combined remediation of heavy metals in soil.

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in Monascus spp.

Gene expression is needed to be conducted in an orthogonal manner and controllable independently from the host's native regulatory system. However, there is a shortage of gene expression regulatory toolboxes that function orthogonally from each other and toward the host. Herein, we developed a strategy based on the mutant library to generate orthogonal gene expression toolboxes. A transcription factor, MaR, located in the Monascus azaphilone biosynthetic gene cluster, was taken as a typical example. Nine DNA-binding residues of MaR were identified by molecular simulation and site-directed mutagenesis. We created five MaR multi-site saturation mutagenesis libraries consisting of 10743 MaR variants on the basis of five cognate promoters. A functional analysis revealed that all five tested promoters were orthogonally regulated by five different MaR variants, respectively. Furthermore, fine gene expression tunability and high signal sensitivity of this toolbox are demonstrated by introducing chemically inducible expression modules, designing synthetic promoter elements, and creating protein-protein interaction between MaRs. This study paves the way for a bottom-up approach to build orthogonal gene expression toolboxes.

Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi.

Random base editing is regarded as a fundamental method for accelerating the genomic evolution in both scientific research and industrial applications. In this study, we designed a modular interaction-based dual base editor (MIDBE) that assembled a DNA helicase and various base editors through dockerin/cohesin-mediated protein-protein interactions, resulting in a self-assembled MIDBE complex capable of editing bases at any locus in the genome. The base editing type of MIDBE can be readily controlled by the induction of cytidine or/and adenine deaminase gene expression. MIDBE exhibited the highest editing efficiency 2.3 × 103 times greater than the native genomic mutation rate. To evaluate the potential of MIDBE in genomic evolution, we developed a removable plasmid-based MIDBE tool, which led to a remarkable 977.1% increase of lovastatin production in Monascus purpureus HJ11. MIDBE represents the first biological tool for generating and accumulating base mutations in Monascus chromosome and also offers a bottom-up strategy for designing the base editor.

Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

Human adenovirus B7 (HAdV-B7) causes severe acute lower respiratory tract infections in children. However, neither the child-specific antivirals or vaccines are available, nor the pathogenesis is clear. Autophagy, as part of innate immunity, plays an important role in resistance to viral infection by degrading the virus and promoting the development of innate and adaptive immunity. This study provided evidence that HAdV-B7 infection induced complete autophagic flux, and the pharmacological induction of autophagy decreased HAdV-B7 replication. In this process, the host protein Bcl2-associated athanogene 3 (BAG3) mediated autophagy to inhibit the replication of HAdV-B7 by binding to the PPSY structural domain of viral protein pVI through its WW structural domain. These findings further our understanding of the host immune response during viral infection and will help to develop broad anti-HAdV therapies.

Activation of glucagon-like peptide-1 receptor in microglia exerts protective effects against sepsis-induced encephalopathy via attenuating endoplasmic reticulum stress-associated inflammation and apoptosis in a mouse model of sepsis.

Sepsis-induced encephalopathy (SAE) is a detrimental complication in patients with severe sepsis, while there is still no effective treatment. Previous studies have elucidated the neuroprotective effects of glucagon-like peptide-1 receptor (GLP-1R) agonists. However, the role of GLP-1R agonists in the pathological process of SAE is unclear. Here, we found that GLP-1R was up-regulated in the microglia of septic mice. The activation of GLP-1R with Liraglutide could inhibit endoplasmic reticulum stress (ER stress) and associated inflammatory response as well as apoptosis triggered by LPS or tunicamycin (TM) in BV2 cells. In vivo experiments confirmed the benefits of Liraglutide in the regulation of microglial activation, ER stress, inflammation, and apoptosis in the hippocampus of septic mice. Additionally, the survival rate and cognitive dysfunction of septic mice were also improved after Liraglutide administration. Mechanically, cAMP/PKA/CREB signaling is involved in the protection of ER stress-induced inflammation and apoptosis in cultured microglial cells under LPS or TM stimulations. In conclusion, we speculated that GLP-1/GLP-1R activation in microglia might be a potential therapeutic target for the treatment of SAE.

[Research on using 3-D fluorescence spectroscopy-wavelet transform-PSO for rapid discrimination of compositions of phytoplankton population].

Three-dimensional fluorescence of 17 red tide algae species that belong to 13 genera of five divisions was measured by fluorescence excitation-emission matrix spectroscopy. And 2-D wavelet db7 was selected to decompose the spectra at different levels to choose the alternative characteristic spectra. Based on the norm reference spectra constructed by cluster analysis, the linear regression model was solved by particle swarm optimization (PSO) algorithm and the discrimination method was established at the division and genus level. Some samples were tested: for single algal samples, and the correct discrimination ratios (CDRs) were 96.1% and 73.6%, respectively; For simulative mixed algal samples, when the dominance were 60%, 75%, 80% and 90% of the total biomass, the CDRs of the dominant algae were 86.7%, 96.9%, 98.7% and 99.4% with the average relative contents of 62.6%, 72.7%, 76.0% and 81.6%, respectively at the division level. And the CDRs were 51.0%, 68.9%, 72.0%, and 78.8% at the genus level, respectively. For 364 actual mixture samples, the CDRs of the dominant species (75%) were 99.4% at the division level and 75.9% at the genus level . For the particular field samples from mesocosm experiment and corrected from Jiaozhou Bay, results showed that the method can be used to realize the identification of red tide algae population and estimate the relative abundance of different classes, especially between diatoms and dinoflagellates.

Systematic Metabolic Engineering for the Production of Azaphilones in Monascus purpureus HJ11.

Fungal azaphilones have attracted considerable interest as they exhibit great potential in food and pharmacological industries. However, there is a severe bottleneck in the low production in wild strains and the ability to genetically engineer azaphilone-producing fungi. Using Monascus azaphilones (MAs) as an example, we demonstrate a systematic metabolic engineering strategy for improving the production of MAs. In this study, Monascus purpureus HJ11 was systematically engineered through a combination of promoter engineering, gene knockout, rate-limiting enzyme overexpression, repression of the competing pathway, enzyme engineering, and metabolic rebalance. The maximum yield and titer of MAs successfully increased to 906 mg/g dry cell weight (DCW) and 14.6 g/L, respectively, 2.6 and 3.7 times higher than those reported in the literature. Our successful model not only offers a practical and efficient way to improve the azaphilone production but also sheds light on the potential of systematic metabolic engineering in nonmodel fungi as a chassis for the production of high-value chemicals.

Simulation and Experimental Study on Roll-Forming Limit of Cup.

Roll forming can improve the material utilization rate and production efficiency of cups with a curved rotary profile, but there is no basis for the determination of forming limit. The DEFORM-3D software was used to simulate the roll forming of cups. The influence of the billet wall thickness and bottom thickness, coefficient of friction, radius of roller, and the fillet radius of the punch on the forming limit was studied, and the damage value and velocity vector were analyzed. The results showed that the forming limit of the billet's wall thickness in roll forming for a cup is about 62%. With the increase of the ratio of the formed cup's wall thickness to the billet's bottom thickness, the forming limit of wall thickness will be slightly reduced. A larger radius of roller, fillet radius of punch, and friction coefficient between punch and billet and a smaller friction coefficient between roller and billet are good for decreasing the damage value and improving the roll-forming limit. According to the numerical simulation results, the roll-forming limit diagram of cups is established, and the accuracy of the forming limit diagram is verified by experiments.