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1.
Curr Microbiol ; 81(6): 161, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38700667

RESUMO

In the wake of rapid industrialization and burgeoning transportation networks, the escalating demand for fossil fuels has accelerated the depletion of finite energy reservoirs, necessitating urgent exploration of sustainable alternatives. To address this, current research is focusing on renewable fuels like second-generation bioethanol from agricultural waste such as sugarcane bagasse. This approach not only circumvents the contentious issue of food-fuel conflicts associated with biofuels but also tackles agricultural waste management. In the present study indigenous yeast strain, Clavispora lusitaniae QG1 (MN592676), was isolated from rotten grapes to ferment xylose sugars present in the hemicellulose content of sugarcane bagasse. To liberate the xylose sugars, dilute acid pretreatment was performed. The highest reducing sugars yield was 1.2% obtained at a temperature of 121 °C for 15 min, a solid-to-liquid ratio of 1:25 (% w/v), and an acid concentration of 1% dilute acid H2SO4 that was significantly higher (P < 0.001) yield obtained under similar conditions at 100 °C for 1 h. The isolated strain was statistically optimized for fermentation process by Plackett-Burman design to achieve the highest ethanol yield. Liberated xylose sugars were completely utilized by Clavispora lusitaniae QG1 (MN592676) and gave 100% ethanol yield. This study optimizes both fermentation process and pretreatment of sugarcane bagasse to maximize bioethanol yield and demonstrates the ability of isolated strain to effectively utilize xylose as a carbon source. The desirable characteristics depicted by strain Clavispora lusitaniae shows its promising utilization in management of industrial waste like sugarcane bagasse by its conversion into renewable biofuels like bioethanol.


Assuntos
Biocombustíveis , Celulose , Etanol , Fermentação , Saccharum , Saccharum/metabolismo , Etanol/metabolismo , Celulose/metabolismo , Gerenciamento de Resíduos/métodos , Agricultura , Xilose/metabolismo , Vitis/microbiologia , Hypocreales/metabolismo
2.
Microb Cell Fact ; 23(1): 133, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38720294

RESUMO

BACKGROUND: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. RESULTS: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-ß-glucosidase (αRßG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRßG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. CONCLUSIONS: αRßG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.


Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Hypocreales/metabolismo , Rutina/farmacologia , Rutina/química , Acremonium , Gencitabina , Dissacarídeos/farmacologia , Dissacarídeos/química
3.
J Appl Microbiol ; 135(5)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38692851

RESUMO

AIMS: Clonostachys rosea is a well-known mycoparasite that has recently been investigated as a bio-based alternative to chemical nematicides for the control of plant-parasitic nematodes. In the search for a promising biocontrol agent, the ability of the C. rosea strain PHP1701 to control the southern root-knot nematode Meloidogyne incognita was tested. METHODS AND RESULTS: Control of M. incognita in vitro and in soil by C. rosea strain PHP1701 was significant and concentration dependent. Small pot greenhouse trials confirmed a significant reduction in tomato root galling compared to the untreated control. In a large greenhouse trial, the control effect was confirmed in early and mid-season. Tomato yield was higher when the strain PHP1701 was applied compared to the untreated M. incognita-infected control. However, the yield of non-M. incognita-infected tomato plants was not reached. A similar reduction in root galling was also observed in a field trial. CONCLUSIONS: The results highlight the potential of this fungal strain as a promising biocontrol agent for root-knot nematode control in greenhouses, especially as part of an integrated pest management approach. We recommend the use of C. rosea strain PHP1701 for short-season crops and/or to reduce M. incognita populations on fallow land before planting the next crop.


Assuntos
Hypocreales , Controle Biológico de Vetores , Doenças das Plantas , Raízes de Plantas , Microbiologia do Solo , Solanum lycopersicum , Tylenchoidea , Solanum lycopersicum/parasitologia , Animais , Tylenchoidea/fisiologia , Raízes de Plantas/parasitologia , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Hypocreales/fisiologia , Solo/parasitologia
4.
Curr Microbiol ; 81(7): 184, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38771325

RESUMO

Agriculture and livestock management practices known as organic farming rely more on internal processes than external inputs. Natural environments depend heavily on diversity, and organic farming incorporates both the stated purpose of fostering diversity as well as the use of diversity as a management tool. A more complete understanding of agriculture in terms of agro-ecology has begun to be questioned by the traditional reductionist approach to the study of agriculture. Therefore it is necessary to be aware more about the significance of microbes in processes including soil growth, plant nourishment, and the eradication of plant disease, pest, and weeds. In this study, fluorescent Pseudomonas strain (EFP56) and Trichoderma harzianum were studied for antifungal and antibacterial activity against four common root rot fungi and four common laboratory bacteria in vitro experiments. Furthermore, soil-borne disease surveillance and nutritional quality of Lagenaria siceraria, fluorescent Pseudomonas strain (EFP56) and Trichoderma harzianum were combined with neem cake and cotton cake to check their efficacy. Through the application of organic soil amendments in combination with biocontrol agents improved the quality of vegetables and their nutritional value by raising their polyphenol, carbohydrate, and protein content as well as enhancing antioxidant scavenging status. The experiments were conducted in pots and in fields to confirm their efficacy rate. The final outcomes also revealed greater induction of defense system, disease lessening and enriched fruit quality. Consortium of neem cake and cotton cake with bio-stimulants can regulate biotic as well as abiotic stress.


Assuntos
Endófitos , Pseudomonas , Microbiologia do Solo , Endófitos/fisiologia , Pseudomonas/fisiologia , Cucurbitaceae/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Hypocreales/fisiologia , Fungos/fisiologia , Fungos/efeitos dos fármacos , Bactérias/classificação , Bactérias/efeitos dos fármacos , Agentes de Controle Biológico , Raízes de Plantas/microbiologia , Antifúngicos/farmacologia , Antifúngicos/metabolismo
5.
Antonie Van Leeuwenhoek ; 117(1): 64, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38565745

RESUMO

Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.


Assuntos
Vesículas Extracelulares , Hypocreales , Trichoderma , Trichoderma/metabolismo , Proteômica
6.
Chemosphere ; 355: 141836, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38561160

RESUMO

The biological synthesis of silver nanoparticles (Ag-NPs) with fungi has shown promising results in antibacterial and antioxidant properties. Fungi generate metabolites (both primary and secondary) and proteins, which aid in the formation of metal nanoparticles as reducing or capping agents. While several studies have been conducted on the biological production of Ag-NPs, the exact mechanisms still need to be clarified. In this study, Ag-NPs are synthesized greenly using an unstudied fungal strain, Sarocladium subulatum AS4D. Three silver salts were used to synthesize the Ag-NPs for the first time, optimized using a cell-free extract (CFE) strategy. Additionally, these NPs were assessed for their antimicrobial and antioxidant properties. Various spectroscopic and microscopy techniques were utilized to confirm Ag-NP formation and analyze their morphology, crystalline properties, functional groups, size, stability, and concentrations. Untargeted metabolomics and proteome disruption were employed to explore the synthesis mechanism. Computational tools were applied to predict metabolite toxicity and antibacterial activity. The study identified 40 fungal metabolites capable of reducing silver ions, with COOH and OH functional groups playing a pivotal role. The silver salt type impacted the NPs' size and stability, with sizes ranging from 40 to 52 nm and zeta potentials from -0.9 to -30.4 mV. Proteome disruption affected size and stability but not shape. Biosynthesized Ag-NPs using protein-free extracts ranged from 55 to 62 nm, and zeta potentials varied from -18 to -27 mV. Molecular docking studies and PASS results found no role for the metabolome in antibacterial activity. This suggests the antibacterial activity comes from Ag-NPs, not capping or reducing agents. Overall, the research affirmed the vital role of specific reducing metabolites in the biosynthesis of Ag-NPs, while proteins derived from biological extracts were found to solely affect their size and stability.


Assuntos
Hypocreales , Nanopartículas Metálicas , Prata , Prata/farmacologia , Prata/química , Antioxidantes/farmacologia , Antioxidantes/química , Simulação de Acoplamento Molecular , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/química , Proteoma , Espectroscopia de Infravermelho com Transformada de Fourier , Antibacterianos/toxicidade , Antibacterianos/química , Extratos Vegetais/química , Testes de Sensibilidade Microbiana
7.
J Agric Food Chem ; 72(15): 8444-8459, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38574108

RESUMO

Cytochrome P450 sterol 14α-demethylase (CYP51) is a key enzyme involved in the sterol biosynthesis pathway and serves as a target for sterol demethylation inhibitors (DMIs). In this study, the 3D structures of three CPY51 paralogues from Calonectria ilicicola (C. ilicicola) were first modeled by AlphaFold2, and molecular docking results showed that CiCYP51A, CiCYP51B, or CiCYP51C proteins individually possessed two active pockets that interacted with DMIs. Our results showed that the three paralogues play important roles in development, pathogenicity, and sensitivity to DMI fungicides. Specifically, CiCYP51A primarily contributed to cell wall integrity maintenance and tolerance to abiotic stresses, and CiCYP51B was implicated in sexual reproduction and virulence, while CiCYP51C exerted negative regulatory effects on sterol 14α-demethylase activity within the ergosterol biosynthetic pathway, revealing its genus-specific function in C. ilicicola. These findings provide valuable insights into developing rational strategies for controlling soybean red crown rot caused by C. ilicicola.


Assuntos
Sistema Enzimático do Citocromo P-450 , Hypocreales , Lanosterol , Lanosterol/metabolismo , Simulação de Acoplamento Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Esteróis , Esterol 14-Desmetilase/química
8.
Food Microbiol ; 121: 104496, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38637067

RESUMO

Phospholipase D plays a critical regulatory role in the pathogenicity of filamentous fungi. However, the molecular mechanism of PLD regulating the pathogenicity of filamentous fungi has not been reported. In this research, the previously constructed TrPLD1 and TrPLD2 (TrPLDs) mutants were used as test strains. Firstly, the function of TrPLDs in Trichothecium roseum was studied. Then, the effects of TrPLDs on the pathogenicity of T. roseum and the quality of the inoculated apples were verified. The results suggested that the deletion of TrPLD1 delayed the spore germination of ΔTrPLD1 and inhibited germ tube elongation by down-regulating the expressions of TrbrlA, TrabaA and TrwetA. By down-regulating the extracellular enzyme-coding gene expressions, ΔTrPLD1 inhibited the degradation of apple fruit cell wall and the change of fatty acid content during infection, reduced the cell membrane permeability and malondialdehyde (MDA) content of apple fruit, thereby maintaining the integrity of fruit cell membrane, and reduced the pathogenicity of ΔTrPLD1 to apple and kept the quality of apple. However, ΔTrPLD2 did not have a significant effect on the infection process of apple fruit by the pathogen.


Assuntos
Hypocreales , Malus , Malus/microbiologia , Frutas/microbiologia , Virulência/genética
9.
J Agric Food Chem ; 72(15): 8742-8748, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38564658

RESUMO

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.


Assuntos
Reagentes de Ligações Cruzadas , Expressão Gênica , Globulinas , Hypocreales , Monofenol Mono-Oxigenase , Proteínas Recombinantes , Proteínas de Soja , Monofenol Mono-Oxigenase/biossíntese , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/isolamento & purificação , Monofenol Mono-Oxigenase/metabolismo , Reagentes de Ligações Cruzadas/isolamento & purificação , Reagentes de Ligações Cruzadas/metabolismo , Hypocreales/classificação , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Hypocreales/metabolismo , Globulinas/química , Globulinas/metabolismo , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Eletroporação , Celulose , Sulfato de Amônio , Cromatografia em Gel , Precipitação Fracionada , Emulsões/química , Emulsões/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Estabilidade Proteica , Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas , Óleos/química , Água/química
10.
J Invertebr Pathol ; 204: 108107, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38614292

RESUMO

The sugarcane aphid, Melanaphis sacchari, is a widely distributed insect that attacks grasses in different genera including Miscanthus, Saccharum, and Sorghum. The invasive aphid superclone was first discovered in the U.S. attacking grain sorghum in Texas in 2013. Since then, it has been found in at least 25 states including Georgia. We conducted a survey of naturally occurring fungal pathogens of sugarcane aphids on five farms in Georgia, and identified a hypocrealean fungus, Akanthomyces dipterigenus, and two entomophthoralean fungi, Neoconidiobolus spp. From 2018 to 2020, fungal activity differed across farms but at one farm both major fungal species, A. dipterigenus and N. thromboides, were found each of the 3 years infecting sugarcane aphids, attacking adults, both alatae and apterae, and nymphs.


Assuntos
Afídeos , Sorghum , Animais , Afídeos/microbiologia , Sorghum/microbiologia , Sorghum/parasitologia , Georgia , Entomophthorales/fisiologia , Hypocreales/fisiologia
11.
J Hazard Mater ; 471: 134299, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38631252

RESUMO

Trichoderma can enhance the metabolism of organophosphate pesticides in plants, but the mechanism is unclear. Here, we performed high-throughput transcriptome sequencing of roots upon Trichoderma asperellum (TM) inoculation and phoxim (P) application in tomato (Solanum lycopersicum L.). A total of 4059 differentially expressed genes (DEGs) were obtained, including 2110 up-regulated and 1949 down-regulated DEGs in P vs TM+P. COG and KOG analysis indicated that DEGs were mainly enriched in signal transduction mechanisms. We then focused on the pesticide detoxification pathway and screened out cytochrome P450 CYP736A12 as a putative gene for functional analysis. We suppressed the expression of CYP736A12 in tomato plants by virus-induced gene silencing and analyzed tissue-specific phoxim residues, oxidative stress markers, glutathione pool, GST activity and related gene expression. Silencing CYP736A12 significantly increased phoxim residue and induced oxidative stress in tomato plants, by attenuating the TM-induced increased activity of antioxidant and detoxification enzymes, redox homeostasis and transcripts of detoxification genes including CYP724B2, GSH1, GSH2, GR, GPX, GST1, GST2, GST3, and ABC. The study revealed a critical mechanism by which TM promotes the metabolism of phoxim in tomato roots, which can be useful for further understanding the Trichoderma-induced xenobiotic detoxification and improving food safety.


Assuntos
Sistema Enzimático do Citocromo P-450 , Compostos Organotiofosforados , Raízes de Plantas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/crescimento & desenvolvimento , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Compostos Organotiofosforados/toxicidade , Compostos Organotiofosforados/metabolismo , Resíduos de Praguicidas/toxicidade , Resíduos de Praguicidas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Hypocreales/metabolismo , Hypocreales/genética
12.
Mol Nutr Food Res ; 68(9): e2300759, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38651284

RESUMO

SCOPE: Tolypocladium sinense is a fungus isolated from Cordyceps. Cordyceps has some medicinal value and is also a daily health care product. This study explores the preventive effects of T. sinense mycelium polysaccharide (TSMP) on high-fat diet-induced obesity and chronic inflammation in mice. METHODS AND RESULTS: Here, the study establishes an obese mouse model induced by high-fat diet. In this study, the mice are administered TSMP daily basis to evaluate its effect on alleviating obesity. The results show that TSMP can significantly inhibit obesity and alleviate dyslipidemia by regulating the expression of lipid metabolism-related genes such as liver kinase B1 (LKB1), phosphorylated AMP-activated protein kinase (pAMPK), peroxisome proliferator activated receptor α (PPARα), fatty acid synthase (FAS), and hydroxymethylglutaryl-CoA reductase (HMGCR) in the liver. TSMP can increase the protein expression of zona occludens-1 (ZO-1), Occludin, and Claudin-1 in the colon, improve the intestinal barrier dysfunction, and reduce the level of serum LPS, thereby reducing the inflammatory response. 16S rDNA sequencing shows that TSMP alters the intestinal microbiota by increasing the relative abundance of Akkermansia, Lactobacillus, and Prevotellaceae_NK3B31_group, while decreasing the relative abundance of Faecalibaculum. CONCLUSION: The findings show that TSMP can inhibit obesity and alleviates obesity-related lipid metabolism disorders, inflammatory responses, and oxidative stress by modulating the gut microbiota and improving intestinal barrier.


Assuntos
Dieta Hiperlipídica , Microbioma Gastrointestinal , Inflamação , Camundongos Endogâmicos C57BL , Micélio , Obesidade , Dieta Hiperlipídica/efeitos adversos , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Masculino , Micélio/química , Inflamação/tratamento farmacológico , Transtornos do Metabolismo dos Lipídeos/tratamento farmacológico , Camundongos , Metabolismo dos Lipídeos/efeitos dos fármacos , Polissacarídeos/farmacologia , Hypocreales , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Polissacarídeos Fúngicos/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo
13.
Proc Natl Acad Sci U S A ; 121(18): e2322567121, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38648472

RESUMO

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.


Assuntos
Celobiose , Celulase , Celulose , Hypocreales , Celobiose/metabolismo , Celulase/metabolismo , Celulase/antagonistas & inibidores , Celulose/metabolismo , Hypocreales/enzimologia , Hypocreales/metabolismo , Imagem Individual de Molécula/métodos , Domínio Catalítico , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química
14.
J Environ Manage ; 358: 120781, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38608570

RESUMO

Transforming global agricultural waste into eco-friendly products like industrial enzymes through bioconversion can help address sustainability challenges aligning with the United Nations' Sustainable Development Goals. Present study explored the production of high-yield food-grade cellulolytic enzymes from Trichoderma reesei MTCC 4876, using a novel media formulation with a combination of waste sorghum grass and cottonseed oil cake (3:1). Optimization of physical and environmental parameters, along with the screening and optimization of media components, led to an upscaled process in a novel 6-L solid-state fermentation (SSF)-packed bed reactor (PBR) with a substrate loading of 200 g. Saturated forced aeration proved crucial, resulting in high fungal biomass (31.15 ± 0.63 mg glucosamine/gm dry fermented substrate) and high yield cellulase (20.64 ± 0.36 FPU/g-ds) and xylanase (16,186 ± 912 IU/g-ds) production at an optimal airflow rate of 0.75 LPM. The PBR exhibited higher productivity than shake flasks for all the enzyme systems. Microfiltration and ultrafiltration of the crude cellulolytic extract achieved 94% and 71% recovery, respectively, with 13.54 FPU/mL activity in the cellulolytic enzyme concentrate. The concentrate displayed stability across wide pH and temperature ranges, with a half-life of 24.5-h at 50 °C. The cellulase concentrate, validated for food-grade safety, complies with permissible limits for potential pathogens, heavy metals, mycotoxins, and pesticide residue. It significantly improved apple juice clarity (94.37 T%) by reducing turbidity (21%) and viscosity (99%) while increasing total reducing sugar release by 63% compared with untreated juice. The study also highlighted the potential use of lignin-rich fermented end residue for fuel pellets within permissible SOx emission limits, offering sustainable biorefinery prospects. Utilizing agro wastes in a controlled bioreactor environment underscores the potential for efficient large-scale cellulase production, enabling integration into food-grade applications and presenting economic benefits to fruit juice industries.


Assuntos
Reatores Biológicos , Fermentação , Sucos de Frutas e Vegetais , Hypocreales , Sorghum , Sorghum/metabolismo , Sucos de Frutas e Vegetais/análise , Celulase/metabolismo , Malus
15.
Microb Cell Fact ; 23(1): 120, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38664812

RESUMO

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.


Assuntos
Esterases , Metionina , Esterases/metabolismo , Esterases/genética , Metionina/metabolismo , Xilanos/metabolismo , Sulfato de Amônio/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Hypocreales/metabolismo , Hypocreales/enzimologia , Hypocreales/genética , Lignina/metabolismo , Acetilação
16.
Sci Rep ; 14(1): 9625, 2024 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-38671155

RESUMO

The filamentous ascomycete Trichoderma reesei, known for its prolific cellulolytic enzyme production, recently also gained attention for its secondary metabolite synthesis. Both processes are intricately influenced by environmental factors like carbon source availability and light exposure. Here, we explore the role of the transcription factor STE12 in regulating metabolic pathways in T. reesei in terms of gene regulation, carbon source utilization and biosynthesis of secondary metabolites. We show that STE12 is involved in regulating cellulase gene expression and growth on carbon sources associated with iron homeostasis. STE12 impacts gene regulation in a light dependent manner on cellulose with modulation of several CAZyme encoding genes as well as genes involved in secondary metabolism. STE12 selectively influences the biosynthesis of the sorbicillinoid trichodimerol, while not affecting the biosynthesis of bisorbibutenolide, which was recently shown to be regulated by the MAPkinase pathway upstream of STE12 in the signaling cascade. We further report on the biosynthesis of dehydroacetic acid (DHAA) in T. reesei, a compound known for its antimicrobial properties, which is subject to regulation by STE12. We conclude, that STE12 exerts functions beyond development and hence contributes to balance the energy distribution between substrate consumption, reproduction and defense.


Assuntos
Carbono , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição , Carbono/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Hypocreales/metabolismo , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Celulose/metabolismo , Celulose/biossíntese , Metabolismo Secundário
17.
ACS Chem Biol ; 19(4): 886-895, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38576157

RESUMO

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer Sarocladium zeae coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare cis-decalin, cytochalasan, and fused 6/15/5 macrocycles. This diversity highlights the extraordinary plasticity of the pyrrocidine biosynthetic gene cluster. From accumulating metabolites, we delineated the scenario of pyrrocidine biosynthesis. The ring A of the decahydrofluorene is installed by PrcB, a membrane-bound cyclizing isomerase, on a PKS-NRPS-derived pyrrolidone precursor. Docking experiments in PrcB allowed us to characterize the active site suggesting a mechanism triggered by arginine-mediated deprotonation at the terminal methyl of the substrate. Next, two integral membrane proteins, PrcD and PrcE, each predicted as a four-helix bundle, perform hydroxylation of the pyrrolidone ring and paracyclophane formation, respectively. Modelization of PrcE highlights a topological homology with vitamin K oxido-reductase and the presence of a disulfide bond. Our results suggest a previously unsuspected coupling mechanism via a transient loss of aromaticity of tyrosine residue to form the strained paracyclophane motif. Finally, the lipocalin-like protein PrcX drives the exo-cycloaddition yielding ring B and C of the decahydrofluorene to afford pyrrocidine A, which is transformed by a reductase PrcI to form pyrrocidine B. These insights will greatly facilitate the microbial production of pyrrocidine analogues by synthetic biology.


Assuntos
Racionalização , Tirosina , Modelos Moleculares , Oxirredutases , Pirrolidinonas/química , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Simulação de Acoplamento Molecular , Hypocreales/química
18.
Microb Cell Fact ; 23(1): 112, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622596

RESUMO

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.


Assuntos
Celulase , Hypocreales , Trichoderma , Celulase/metabolismo , Lactose/metabolismo , Reprodutibilidade dos Testes , Biotecnologia , Trichoderma/metabolismo
19.
Microbiol Spectr ; 12(4): e0316523, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38441469

RESUMO

Trichoderma species are known for their mycoparasitic activity against phytopathogenic fungi that cause significant economic losses in agriculture. During mycoparasitism, Trichoderma spp. recognize molecules produced by the host fungus and release secondary metabolites and hydrolytic enzymes to kill and degrade the host's cell wall. Here, we explored the participation of the Trichoderma atroviride RNAi machinery in the interaction with six phytopathogenic fungi of economic importance. We determined that both Argonaute-3 and Dicer-2 play an essential role during mycoparasitism. Using an RNA-Seq approach, we identified that perception, detox, and cell wall degradation depend on the T. atroviride-RNAi when interacting with Alternaria alternata, Rhizoctonia solani AG2, and R. solani AG5. Furthermore, we constructed a gene co-expression network that provides evidence of two gene modules regulated by RNAi, which play crucial roles in essential processes during mycoparasitism. In addition, based on small RNA-seq, we conclude that siRNAs regulate amino acid and carbon metabolism and communication during the Trichoderma-host interaction. Interestingly, our data suggest that siRNAs might regulate allorecognition (het) and transport genes in a cross-species manner. Thus, these results reveal a fine-tuned regulation in T. atroviride dependent on siRNAs that is essential during the biocontrol of phytopathogenic fungi, showing a greater complexity of this process than previously established.IMPORTANCEThere is an increasing need for plant disease control without chemical pesticides to avoid environmental pollution and resistance, and the health risks associated with the application of pesticides are increasing. Employing Trichoderma species in agriculture to control fungal diseases is an alternative plant protection strategy that overcomes these issues without utilizing chemical fungicides. Therefore, understanding the biocontrol mechanisms used by Trichoderma species to antagonize other fungi is critical. Although there has been extensive research about the mechanisms involved in the mycoparasitic capability of Trichoderma species, there are still unsolved questions related to how Trichoderma regulates recognition, attack, and defense mechanisms during interaction with a fungal host. In this work, we report that the Argonaute and Dicer components of the RNAi machinery and the small RNAs they process are essential for gene regulation during mycoparasitism by Trichoderma atroviride.


Assuntos
Hypocreales , Praguicidas , Plantas , Comunicação , Regulação Fúngica da Expressão Gênica
20.
J Invertebr Pathol ; 204: 108078, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38438078

RESUMO

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia, Italy and the rest of Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P, spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P, spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave way for testing EPF isolates against P, spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.


Assuntos
Hemípteros , Ninfa , Controle Biológico de Vetores , Animais , Ninfa/microbiologia , Ninfa/crescimento & desenvolvimento , Controle Biológico de Vetores/métodos , Hemípteros/microbiologia , Beauveria/patogenicidade , Beauveria/fisiologia , Insetos Vetores/microbiologia , Fusarium , Itália , Xylella/fisiologia , Hypocreales/fisiologia , Hypocreales/patogenicidade
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