Promoter screening and identification for metabolic regulation in Acremonium chrysogenum.

Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter PAngpdA . Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high-yielding strains AG-6, AG-18, and AG-41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac-∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression.

A systematic analysis of ARM genes revealed that GhARM144 regulates the resistance against Verticillium dahliae via interaction with GhOSM34.

Proteins of the armadillo repeat gene family play important roles in plant pathogen response. Here, 169 armadillo (ARM) genes were identified in upland cotton (Gossypium hirsutum). Phylogenetic analysis grouped these into 11 subfamilies, with conserved protein structures within each subfamily. The results signify that the expansion of the gene family occurred via whole genome duplication and dispersed duplication. Expression profiling and network analysis suggest that GhARM144 may regulate cotton resistance to Verticillium dahliae. GhARM144 was upregulated in roots by V. dahliae infection or salicylic acid treatment. This upregulation indicates a negative regulatory role of GhARM144' in the cotton immune responses, potentially by manipulating salicylic acid biosynthesis. Protein interaction studies found that GhARM144 associates with an osmotin-like protein, GhOSM34, at the plasma membrane. Silencing GhOSM34 reduced the resistance to V. dahliae, suggesting it may play a positive regulatory role. The results demonstrate that GhARM144 modulates cotton immunity through interaction with GhOSM34 and salicylic acid signalling. Further study of these proteins may yield insights into disease resistance mechanisms in cotton and other plants.

Acremonium sp. diglycosidase-aid chemical diversification: valorization of industry by-products.

The fungal diglycosidase α-rhamnosyl-ß-glucosidase I (αRßG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRßG I in valorizing glycerol and agricultural by-products. KEYPOINTS: • αRßG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. • The glycosylation of aliphatic polyalcohols by the αRßG I resulted in products bearing a single rutinose moiety. • αRßG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield).

The secreted feruloyl esterase of Verticillium dahliae modulates host immunity via degradation of GhDFR.

Feruloyl esterase (ferulic acid esterase, FAE) is an essential component of many biological processes in both eukaryotes and prokaryotes. This research aimed to investigate the role of FAE and its regulation mechanism in plant immunity. We identified a secreted feruloyl esterase VdFAE from the hemibiotrophic plant pathogen Verticillium dahliae. VdFAE acted as an important virulence factor during V. dahliae infection, and triggered plant defence responses, including cell death in Nicotiana benthamiana. Deletion of VdFAE led to a decrease in the degradation of ethyl ferulate. VdFAE interacted with Gossypium hirsutum protein dihydroflavanol 4-reductase (GhDFR), a positive regulator in plant innate immunity, and promoted the degradation of GhDFR. Furthermore, silencing of GhDFR led to reduced resistance of cotton plants against V. dahliae. The results suggested a fungal virulence strategy in which a fungal pathogen secretes FAE to interact with host DFR and interfere with plant immunity, thereby promoting infection.

A Berberine Bridge Enzyme-like Oxidase Mediates the Cage-like Acresorbicillinol C Biosynthesis.

Oxosorbicillinol and cage-like acresorbicillinol C are bioactive sorbicillinoids produced by Acremonium chrysogenum. We found that a berberine bridge enzyme-like oxidase AcsorD was responsible for their biosynthesis by gene deletion and heterologous expression. AcsorD catalyzed oxidation of sorbicillinol to form oxosorbicillinol in in vitro assays, which was successively condensed with sorbicillinol to form acresorbicillinol C spontaneously. Finally, site-directed mutation revealed that Tyr525 was the key residue in the catalysis of the oxidation reaction and unlocking cage-like acresorbicillinol C production.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

CRISPR-based platforms for the specific and dual detection of defoliating/nondefoliating strains of Verticillium dahliae.

BACKGROUND: Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt disease on more than 400 plant species worldwide. Because of its broad host range and its ability to survive long term in the soil, there are few effective control measures for V. dahliae once it has become established. Accurate, sensitive, and rapid detection of V. dahliae is crucial for limiting pathogen entry into new regional environments and early management of Verticillium wilt. RESULTS: In this study, we developed a method to detect V. dahliae based on recombinase polymerase amplification (RPA) and CRISPR/Cas technology and used fluorescence and lateral flow test strips to monitor the outcomes. Through the establishment and optimization of RPA-CRISPR/Cas13a detection, the sensitivity of the fluorescence method was 1 am for genomic DNA (gDNA) within 20 min, whereas the sensitivity of the lateral flow strip method was 100 am for gDNA in 30 min. The field applicability of RPA-CRISPR/Cas13a was also validated by the detection of V. dahliae on smoke trees (Cotinus coggygria) in Xiangshan Park, Beijing, China. Finally, diplex detection for defoliating and nondefoliating pathotypes of V. dahliae was established by combining CRISPR-Cas12a/Cas13a with specific target genes. CONCLUSION: Taken together, this study achieved rapid, sensitive, and accurate detection of V. dahliae and the differentiation of defoliating and nondefoliating pathotypes and provides potential for field-deployable diagnostic tools for rapid and ultrasensitive detection. © 2023 Society of Chemical Industry.

Successful treatment of peritonitis caused by Acremonium species without catheter removal: Case report and literature review.

INTRODUCTION: It is a rare case of continuous ambulatory peritoneal dialysis-related peritonitis associated with Acremonium spp infection. CASE PRESENTATION: Symptoms of Acremonium infection peritonitis are hidden and atypical, leucocytes in ascites are moderately elevated, and general bacterial culture difficulty obtains positive results. In this report, a patient with peritoneal dialysis-related peritonitis caused by Acremonium species was successfully treated without catheter removal in our hospital. The organism species was cultured from a catheter and PD effluent fluid. The patient's peritonitis did not relapse within 6 months. CONCLUSIONS: Once a patient on peritoneal dialysis was infected with fungal peritonitis, the outcome was usually to remove the tube and stop peritoneal dialysis. In this case, our experience is that using a catheter-salvage therapy method, we can successfully cure PD-related peritonitis associated with Acremonium sp.

Identification of a (+)-cubenene synthase from filamentous fungi Acremonium chrysogenum.

Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis.

Influence of ultraviolet and chemical treatment on the biodegradation of low-density polyethylene and high-density polyethylene by Cephalosporium strain.

In the present work, the potential of Cephalosporium strain in degrading the pre-treated (ultraviolet irradiation followed by nitric acid treatment) low-density polyethylene and high-density polyethylene films was investigated. Our observations revealed a significant weight reduction of 24.53 ± 0.73% and 18.22 ± 0.31% in pre-treated low-density polyethylene and high-density polyethylene films respectively, after 56 days of incubation with the Cephalosporium strain. Changes in the physicochemical properties of the mineral salt medium (MSM) were studied to assess the extent of biodegradation. The pH of the MSM decreased gradually during the incubation period, whereas its total dissolved solids and conductivity values increased steadily. Fourier transform infrared spectroscopy (FTIR) indicated the formation of hydroxyl and C = C groups in biodegraded low-density polyethylene films, while in the case of biodegraded high-density polyethylene films it indicated the [Formula: see text]CH2 stretching. Furthermore, the thermogravimetric analysis (TGA) revealed an enhancement in the thermal stabilities of both the LDPE and HDPE films post the biodegradation. Modifications in the polymer surface morphologies after UV irradiation, chemical treatment, and biodegradation steps were visualized via scanning electron microscopy (SEM) analysis. All our observations confirm the ability of the Cephalosporium strain in biodegrading the pre-treated LDPE and HDPE films.

A Multilocus Phylogeny Places Hymenula cerealis (syn. Cephalosporium gramineum) in the Helotiales, Leotiomycetes.

The soilborne fungus Hymenula cerealis causes Cephalosporium stripe, a vascular wilt disease of wheat and other grasses in the United States and other wheat-producing countries where winter wheat is subjected to snow cover and frozen soil. No sexual stage is known for H. cerealis, and consequently, its phylogenetic position relative to other fungi has been difficult to establish. The purpose of this study was to conduct a multilocus sequence analysis to determine the phylogenetic position of H. cerealis. Sequence data for five genes, the internal transcribed spacer (ITS), partial large subunit nrDNA (LSU), partial RNA polymerase II second largest subunit region (RPB2), ß-tubulin gene, and translation elongation factor 1-α (TEF1-α), from a diverse set of C. gramineum isolates and other related fungi was obtained from GenBank or directly from isolates in the Murray lab and used to construct maximum-likelihood and Bayesian trees. Based on phylogenetic analysis of the single LSU and ß-tubulin genes, Cephalosporium gramineum is closely related to the Drepanopezizaceae and Ploettnerulaceae of Helotiales. Based on analyses of the DNA sequence of the ITS, RPB2, and TEF1-α genes, as well as the combined five-gene data set, C. gramineum belongs to the family Drepanopezizaceae, which is a sister taxon to the Ploettnerulaceae, and formed a well-supported clade (MLBP/BIPP = 95%/100%). In conclusion, H. cerealis belongs to the Helotiales, Leotiomycetes.

Seed Infection Rate, but Not Pathogen Titer, Positively Correlates with Disease Index of Cephalosporium Stripe in Winter Wheat.

Cephalosporium gramineum survives primarily in colonized plant residue but is also transmitted by seed at a low frequency. The purpose of this study was to correlate disease intensity in the field with percentage of infected seed and amount of pathogen DNA using a high-throughput PCR method. Field-grown seed of three wheat cultivars was collected over 4 years from plots with a known disease index. The culture-based seed infection rate was determined by isolation of C. gramineum from 2,016 seeds per seed lot. DNA of 380 seeds from each seed lot was extracted individually, and a PCR assay with a fluorescent-labeled forward primer for detecting C. gramineum was performed on each seed. C. gramineum was isolated from 0.12% of the seed on average (range 0 to 0.74%), whereas it was detected in 3.7% on average (range 1.3 to 7.6%) using PCR detection. The single-seed PCR assay was more sensitive than either the culture-based method or conventional PCR. DNA of 674 seeds that tested positive by this PCR was quantified using a real-time PCR with newly designed primers for the amount of pathogen per seed. Seed contained 0.017 to 77.1 pg/seed of C. gramineum DNA (mean 3.0 pg/seed). Disease index was positively correlated with seed infection rate but not with pathogen titer in seed. This fluorescent-labeled PCR, along with quantitative PCR, improved our understanding of seed transmission of C. gramineum in wheat.

Anti-Inflammatory and Proangiogenic Metabolites from the Hadal Trench-Derived Fungus Acremonium dichromosporum YP-213.

Four new compounds, including two ascochlorin-type meroterpenoids acremocholrins A (1) and B (2), one pyridone alkaloid acremopyridone A (7), and one cyclopentenone derivative acremoketene A (12), together with eight known compounds (3-6 and 8-11), were isolated and identified from the hadal trench-derived fungus Acremonium dichromosporum YP-213. Their structures were determined with a detailed spectroscopic analysis of NMR and MS data, NOE analysis, octant rule and quantum chemical calculations of ECD, and NMR (with DP4+ probability analysis). Among the compounds, 7 represent a novel scaffold derived from a pyridone alkaloid by cleavage of the C-16-C-17 bond following oxidation to give a ketone. Compounds 9, 11, and 12 showed potent in vivo anti-inflammatory activity in transgenic zebrafish, while compound 8 exhibited significant proangiogenic activity in transgenic zebrafish.

VdPT1 Encoding a Neutral Trehalase of Verticillium dahliae Is Required for Growth and Virulence of the Pathogen.

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain.

The addition of exogenous polyamines increases the production of antibiotic cephalosporin C (CPC) in Acremonium chrysogenum high-yielding (HY) strain during fermentation on a complex medium. However, the molecular basis of this phenomenon is still unknown. In the current study, we developed a special synthetic medium on which we revealed the opposite effect of polyamines. The addition of 1,3-diaminopropane resulted in an increase in the yield of CPC by 12-15%. However, the addition of spermidine resulted in a decrease in the yield of CPC by 14-15% and accumulation of its metabolic pathway precursor, deacetylcephalosporin C (DAC); the total amount of cephems (DAC and CPC) was the same as after the addition of DAP. This indicates that spermidine, but not 1,3-diaminopropane, affects the final stage of CPC biosynthesis, associated with the acetylation of its precursor. In both cases, upregulation of biosynthetic genes from beta-lactam BGCs occurred at the same level as compared to the control; expression of transport genes was at the control level. The opposite effect may be due to the fact that N1-acetylation is much more efficient during spermidine catabolism than for 1,3-diaminopropane. The addition of spermidine, but not 1,3-diaminopropane, depleted the pool of acetyl coenzyme A by more than two-fold compared to control, which could lead to the accumulation of DAC.

Acremonium (Sarocladium) periprosthetic joint infection: case report, literature review, and proposed antifungal regimen.

BACKGROUND: Fungal periprosthetic joint infections are rare. Acremonium osteoarticular infections are scarcely reported. Variable susceptibility to antifungal agents have been reported and optimal pharmacotherapy has yet to be established. Here we illustrate an Acremonium osteoarticular infection involving a prosthetic joint and present an antifungal regimen that had led to treatment success. CASE PRESENTATION: A 60-year-old female with a body mass index (BMI) of 40 had left total knee arthroplasty done in 2012 with a cementless implant for knee osteoarthritis. In 2019, the patient had asymptomatic, progressive osteolysis with fracture and migration of the femoral component warranting replacement. Eleven months later, the patient developed significant pain, redness, and swelling in the left leg and knee concerning for periprosthetic joint infection that failed outpatient antibiotic treatment. Further investigation revealed infection by Acremonium species. A revision of the joint was successfully completed, and the patient was placed on voriconazole for one year. Subsequent cultures did not yield any fungal growth. CONCLUSION: While an optimal antifungal regimen for periprosthetic joint infections has not been well established, voriconazole is a relatively safe and effective agent that can be used as a long-term therapy. With variable susceptibility testing in reported isolates, individualized antifungal susceptibility should be used to guide therapy for Acremonium infections.

Marine fungi showing multifunctional activity against human pathogenic microbes and cancer.

Multifunctional drugs have shown great promise in biomedicine. Organisms with antimicrobial and anticancer activity in combination with antioxidant activity need further research. The Red Sea and the Arabian Gulf coasts were randomly sampled to find fungi with multifunctional activity. One hundred strains (98 fungi and 2 lichenized forms) were isolated from 15 locations. One-third of the isolates inhibited clinical bacterial (Staphylococcus aureus, Bacillus subtilis, Vibrio cholerae, Salmonella typhi, S. paratyphi) and fungal pathogens (Talaromycets marneffei, Malassezia globose, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus) and four cancer cell lines (Hep G2 liver, A-549 lung, A-431skin, MCF 7 breast cancer). Bacterial and cancer inhibition was often accompanied by a high antioxidant activity, as indicated by the principal component analysis (PCA). PCA also indicated that fungal and bacterial pathogens appeared to be inhibited mostly by different marine fungal isolates. Strains with multifunctional activity were found more from the Rea Sea than from the Arabian Gulf coasts. The highest potential for multifunctional drugs were observed for Acremonium sp., Acrocalymma sp., Acrocalymma africana, Acrocalymma medicaginis (activity reported for the first time), Aspergillus sp. Cladosporium oxysporum, Emericellopsis alkaline, Microdochium sp., and Phomopsis glabrae. Lung, skin, and breast cancers were inhibited 85%-97% by Acremonium sp, while most of the isolates showed low inhibition (ca 20%). The highest antifungal activity was observed for Acremonium sp., Diaporthe hubeiensis, Lasiodiplodia theobromae, and Nannizia gypsea. One Acremonium sp. is of particular interest to offer a multifunctional drug; it displayed both antifungal and antibacterial activity combined with high antioxidant activity (DPPH scavenging 97%). A. medicaginis displayed combined antibacterial, anticancer, and antioxidant activity being of high interest. Several genera and some species included strains with both high and low biological activities pointing out the need to study several isolates to find the most efficient strains for biomedical applications.

Acremonium keratitis: Risk factors, clinical characteristics, management, and outcome in 65 cases.

Purpose: To study the risk factors, clinical presentation, management options, and outcomes in cases of culture-proven Acremonium keratitis. Methods: Medical and microbiology records of culture-proven Acremonium keratitis from Jan 2007 to Dec 2019 at a tertiary eye care center were reviewed. Details of clinical findings on each visit and operating notes were reviewed from the medical records. All cases were subjected to corneal scraping at the first visit for microbiological investigation consisting of direct smear examination and culture. Topical natamycin 5% was the mainstay of medical treatment. Surgical treatment was considered for nonresponding patients. Results: During the 13-year study period, 65 cases of culture-proven Acremonium keratitis were identified out of 1605 cases of fungal keratitis. Trauma was the most common predisposing factor in 32 cases (49.2%). The average area of the corneal stromal infiltrate was 24.8 mm2 at the initial presentation. Hypopyon at the time of presentation was evident in 28 (43.1%) cases. Staphylococcus spp. was the most common (n = 22, 33.8%) organism coexistent with Acremonium. Direct microscopy of corneal scraping was positive for fungal filaments in 57/65 (87.6%) cases. Medical management alone was given in 44 patients (67.6%). Age (>50 years) and treatment delay (>15 days) were found to be independent risk factors for the poor final visual outcome (VA <20/60). Conclusion: When treated early, Acremonium keratitis responds well to medical therapy with currently available topical antifungals. However, advanced and nonresponding cases require surgical intervention for resolution of the infection.