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1.
World J Microbiol Biotechnol ; 35(7): 109, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280382

RESUMO

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31 mg/l and no ST was detected in the 50 l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Equinocandinas/biossíntese , Equinocandinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Policetídeo Sintases/genética , Esterigmatocistina/biossíntese , Agrobacterium/genética , Anidulafungina , Antifúngicos , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , DNA Fúngico/isolamento & purificação , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Metabolismo Secundário/genética , Transformação Genética
2.
Curr Microbiol ; 76(6): 673-677, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941538

RESUMO

Pathogenic fungi, as an increasing global threat to human health, represent a sizable risk. However, significant attention should also be paid to the yeast biofilms. One promising strategy for combating resistant microbes, as well as fungal biofilms, is to extend the lifespan and efficacy of our currently employed drugs by using combination therapy. Since the application of combined therapy of fungal infections is currently accepted, we have decided to verify the efficacy of derivative H in combination with fluconazole on C. albicans biofilm. The main advantage of synergy over monotherapy lies in reducing or even completely preventing the induction of resistance of fungal cells. We have decided to verify the derivative H (1,4-dihydropyridine-2,3,5-tricarboxylate), an intermediate of nilvadipine synthesis, in the resistance of C. albicans to fluconazole. Therefore, we have focused on the influence of derivative H on the gene expression of the main C. albicans adhesin (ALS3), which is important for the tissue colonization during the infection process. Our results show that the newly synthesized derivative H had an impact on biofilm eradication. The effect of biofilm diminution could, therefore, be explained as derivative H preventing the adherence of C. albicans cells. This study supports even more the attractiveness of this substance as a potential agent that could be used in synergy with commonly used azoles to treat various fungal infections.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Sinergismo Farmacológico , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Adesão Celular/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana
3.
Molecules ; 24(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991754

RESUMO

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.


Assuntos
Proteínas Fúngicas , Expressão Gênica , Pichia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
4.
World J Microbiol Biotechnol ; 35(4): 54, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900052

RESUMO

Filamentous fungi are important microorganisms used in industrial production of proteins and enzymes. Among these organisms, Trichoderma reesei, Aspergilli, and more recently Myceliophthora thermophile are the most widely used and promising ones which have powerful protein secretion capability. In recent years, there have been tremendous achievements in understanding the molecular mechanisms of the secretory pathways in filamentous fungi. The acquired pieces of knowledge can be harnessed to enhance protein production in filamentous fungi with assistance of state-of-the-art genetic engineering techniques.


Assuntos
Proteínas Fúngicas/biossíntese , Fungos/metabolismo , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Aspergillus/metabolismo , Códon , Proteínas Fúngicas/genética , Fungos/genética , Fungos/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Glicosilação , Peptídeos/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Saccharomycetales/metabolismo , Via Secretória/genética , Trichoderma/metabolismo
5.
Proc Natl Acad Sci U S A ; 116(15): 7409-7418, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30902897

RESUMO

The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.


Assuntos
Agaricales , Bases de Dados de Ácidos Nucleicos , Carpóforos , Proteínas Fúngicas , Genes Fúngicos , Transcriptoma/fisiologia , Agaricales/genética , Agaricales/crescimento & desenvolvimento , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia
6.
Org Lett ; 21(7): 2204-2208, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30892050

RESUMO

Asymmetric reduction of hydroxynaphthoquinones to secondary metabolites, (3 S,4 R)-3,4,8- and (2 S,4 R)-2,4,8-trihydroxy-1-tetralone, a putative biosynthetic diketo intermediate and a probable natural analogue, (3 S,4 R)-7-acetyl-3,4,8-trihydroxy-6-methyl-3,4-dihydronaphthalene-1(2 H)-one, using NADPH-dependent tetrahydroxynaphthalene reductase (T4HNR) of Magnaporthe grisea is described. This work implies the involvement of T4HNR or related enzymes during the (bio)synthesis of other dihydroarenediols by reduction of the hydroxynaphthoquinone scaffold containing substrates.


Assuntos
Álcoois/síntese química , Proteínas Fúngicas/biossíntese , Magnaporthe/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/biossíntese , Proteínas Fúngicas/química , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química
7.
Int J Biol Macromol ; 127: 683-692, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30703426

RESUMO

A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.


Assuntos
Proteínas Fúngicas , Maltose/química , Pichia , Amido/química , alfa-Amilases , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trissacarídeos/química , alfa-Amilases/biossíntese , alfa-Amilases/química , alfa-Amilases/genética
8.
Appl Microbiol Biotechnol ; 103(7): 3061-3071, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30783720

RESUMO

A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.


Assuntos
Técnicas Biossensoriais , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Pleurotus/enzimologia , Ácidos Cafeicos/metabolismo , Clonagem Molecular , Meios de Cultura/química , Enzimas Imobilizadas/biossíntese , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Lacase/genética , Levodopa/metabolismo , Oxirredução , Pichia/genética , Poliestirenos , Proteínas Recombinantes de Fusão/biossíntese
9.
Bioprocess Biosyst Eng ; 42(5): 829-838, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30739160

RESUMO

In the present study, it was presented a strategy to maximize the cutinase production by solid-state fermentation from different microorganisms and substrates. The best results were observed using Fusarium verticillioides, rice bran being the main substrate. Maximum yield of cutinase obtained by the strain was 16.22 U/g. For concentration, ethanol precipitation was used, and the purification factor was 2.4. The optimum temperature and pH for enzyme activity were 35 °C and 6.5, respectively. The enzyme was stable at a wide range of temperature and at all pH values tested. The concentrated cutinase was used as an adjuvant in a formulation containing cutinase + bioherbicide. The use of enzyme increased the efficiency of bioherbicide, since cutinase was responsible to remove/degrade the cutin that recovery the weed leaves and difficult the bioherbicide absorption. Cutinase showed to be a promising product to be used in formulation of bioherbicides.


Assuntos
Hidrolases de Éster Carboxílico , Proteínas Fúngicas , Fusarium/enzimologia , Herbicidas/metabolismo , Controle Biológico de Vetores , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Herbicidas/química , Concentração de Íons de Hidrogênio , Oryza/química
10.
Bioprocess Biosyst Eng ; 42(5): 753-761, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30805716

RESUMO

Kojic acid is a kind of secondary metabolites, whose biosynthesis pathway remains unclear to date. It is produced industrially by microbial fermentation, and thus, mutagenesis breeding still plays a vital role for obtaining strains with high kojic acid production. The starting strain KA-11 isolated from mildewed fruits was identified as Aspergillus oryzae and then subjected to a combined mutagenesis program including microwave mutagenesis, UV irradiation, heat-LiCl, atmospheric, and room temperature plasma (ARTP). The kojic acid production was increased by 47.0%, 87.1%, 126.2%, and 292.3% compared with the starting strain KA-11 after each mutagenesis stage. A mutant strain AR-47 with kojic acid production of 96.5 g/L in flask-shake fermentation was finally obtained. The fermentation time also decreased from 7 to 5 days. Real-time quantitative PCR was used to determine the transcriptional expression levels of genes that may be relevant to kojic acid biosynthesis, including kojA, kojR, kojT, AO090113000141, AO090113000143, AO090113000145, nrtA, and laeA. The results showed that the transcriptional expression levels of all these genes in high yield strain AR-47 had increased compared with the starting strain KA-11.


Assuntos
Aspergillus oryzae , Proteínas Fúngicas , Mutagênese , Pironas/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética
11.
Int J Mol Sci ; 20(1)2019 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-30621365

RESUMO

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.


Assuntos
Aldeído Redutase/química , Aldeído Redutase/metabolismo , Chaetomium/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Coenzimas/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Temperatura Ambiente
12.
Bioprocess Biosyst Eng ; 42(4): 567-574, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30652220

RESUMO

Chemical composition and physical structure of solid substrate have significantly impacts on fermentation performance. The aqueous ammonia was used to pretreat rice straw. Furthermore, the feasibility of pretreatment to improve laccase production was also evaluated in terms of the enzymatic digestibility, chemical structure, physical structure, and laccase production. The results showed that aqueous ammonia pretreatment could modify chemical compositions, destroy rigid structure of the lignocellulosic substrate, increase enzymatic digestibility and change water state, which were beneficial to facilitate the fungus growth and nutrition utilization. Pretreatment of lignocellulosic substrate with aqueous ammonia at 80 °C gave the best effect on laccase production, yielding 172.74 U/g laccase at 14 days, which was 3.4 times higher than that of the control. The aqueous ammonia pretreatment could alternate the physicochemical characteristics of lignocellulosic substrate, resulting in the improved laccase production, which was a promising method that might be explored in solid-state fermentation.


Assuntos
Basidiomycota/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Lignina , Oryza/química , Fermentação , Hidrólise , Lignina/química , Lignina/metabolismo
13.
Int J Biol Macromol ; 125: 955-961, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30576739

RESUMO

Asparaginase catalyzes the conversion of asparagine into aspartic acid and ammonia. The enzyme has various industrial applications and it is considered as an anticancer drug for treatment of certain leukemias. In the current study, production of asparaginase was investigated by Yarrowia lipolytica as well as optimized its production and determined its molecular characteristics by in silico analysis. Y. lipolytica DSM3286 produced 17.14 U/ml of asparaginase in flask culture. Optimization of asparaginase production was done by response surface methodology and the enzyme production increases up to 102.85 U/ml. The enzyme production reached 210 U/ml in a bioreactor which is 12-fold more than flask culture containing non-optimized medium. Asparaginase gene of Y. lipolytica was identified and isolated on the basis of comparison with asparaginase gene sequences of other microorganisms. The gene has 981 nucleotides and its protein has 326 amino acids. According to in silico analysis, the secondary structure of the enzyme is composed of 9 α-helixes and 11 ß-sheets. Y. lipolytica produces type II asparaginase with high affinity for asparagine which is a suitable eukaryotic asparaginase for treatment of hematopoietic cancers. Hence, Y. lipolytica could be recommended as a new eukaryotic microbial source for the production of this important therapeutic enzyme.


Assuntos
Antineoplásicos/química , Asparaginase/química , Proteínas Fúngicas/química , Microbiologia Industrial/métodos , Yarrowia/enzimologia , Sequência de Aminoácidos , Amônia/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Asparaginase/biossíntese , Asparaginase/isolamento & purificação , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Reatores Biológicos , Meios de Cultura/química , Meios de Cultura/farmacologia , Análise Fatorial , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Yarrowia/química , Yarrowia/efeitos dos fármacos
14.
Bioprocess Biosyst Eng ; 42(3): 499-512, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30536123

RESUMO

Repeated batch semi-solid fermentation (sSF) process using wheat straw substrate and fungal growth of Ganoderma lucidum on solid substrate was studied for production of laccase. pH showed significant effect on laccase production. Highest laccase activity with pH controlled to 5.0 in batch sSF was 15257.2 ± 353.4 U L- 1 on 9th day. In repeated batch process at pH 5.0, insoluble biomass substrate and fungal growth were reused after liquid part of medium was replaced with glucose, ammonium phosphate (best nitrogen source) and combined glucose and ammonium phosphate solution separately. Refilled to 80% w v- 1 of initial soluble sugar of first batch resulted in highest laccase production with peak activity after 4 days from replacement. Production of enzyme increased from 15257.2 U L- 1 in first batch to cumulative 90164.4 U L- 1 in 29 days after six repeated batches, productivity increased from 1680.2 to 3110.3 U L- 1 day- 1 (∼ 1.9 times) due to reductions in inhibitory effects and time required for fungal growth. Utilization of wheat straw in repeated batch sSF was supported by composition analysis and morphological changes (scanning electron microscopy) of substrate. Economic production of laccase using agricultural residues in repeated batch sSF could be possible.


Assuntos
Biomassa , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Reishi/crescimento & desenvolvimento , Triticum/química , Técnicas de Cultura Celular por Lotes
15.
Mol Cell ; 72(6): 1013-1020.e6, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576652

RESUMO

Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.


Assuntos
Caulobacter crescentus/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminopeptidases/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Caulobacter crescentus/genética , Linhagem Celular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Fúngico/química , RNA Fúngico/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Ribossomos/química , Ribossomos/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
16.
World J Microbiol Biotechnol ; 34(11): 160, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341455

RESUMO

Pleurotus tuoliensis is a valuable, rare and edible mushroom that is been commercially cultivated and is rapidly developing in China markets. Low temperatures are required to induces primordia initiation for the successful production of fruiting bodies (basidiomes) during commercial cultivation. In this work, we investigated the enzymatic activities and performed transcription profiling analysis of enzymatic genes under different low temperature conditions. The results suggest that the enzymatic activities and transcription levels decrease or increase significantly at 4 and 13 °C. Lacc10 and mnp6 seems to play a dominant role during nutrition growth. Furthermore, the expression of laccase and peroxidase genes was highly correlated to the detected extracellular enzymatic activity. Cold stress genes expression profiles were upregulated under 4 °C/13 °C (3 days), while only the Hsp70 gene was downregulated (at the stage of fruiting bodies production) at 13 °C (12 days). Our results showed that the transcriptional regulation of laccase and ligninolytic peroxidase genes plays an important role in the fruiting bodies of Bailinggu under low temperature induction (4 °C). Induction at low temperatures was a highly important cultivation condition in Bailinggu.


Assuntos
Temperatura Baixa , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Pleurotus/enzimologia , Pleurotus/genética , Catalase/biossíntese , Catalase/genética , Catecol Oxidase/biossíntese , Catecol Oxidase/genética , China , Ensaios Enzimáticos , Perfilação da Expressão Gênica , Lacase/biossíntese , Lacase/genética , Peroxidase/biossíntese , Peroxidase/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Transcriptoma
17.
World J Microbiol Biotechnol ; 34(11): 162, 2018 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-30368630

RESUMO

This study was conducted to report the richness of endophytic Penicillium and Talaromyces species isolated from Tillandsia catimbauensis, a bromeliad endemic in the Brazilian tropical dry forest (Caatinga), to verify their ability to produce the enzyme L-asparaginase and to partially optimise the production of biomass and L-asparaginase of the best enzyme producer. A total of 184 endophytes were isolated, of which 52 (29%) were identified through morphological and phylogenetic analysis using ß-tubulin sequences into nine putative species, four in Penicillium and five in Talaromyces. Talaromyces diversus and T. cf. cecidicola were the most frequent taxa. Among the 20 endophytic isolates selected for L-asparaginase production, 10 had the potential to produce the enzyme (0.50-2.30 U/g), especially T. cf. cecidicola URM 7826 (2.30 U/g) and Penicillium sp. 4 URM 7827 (1.28 U/g). As T. cf. cecidicola URM 7826 exhibited significant ability to produce the enzyme, it was selected for the partial optimisation of biomass and L-asparaginase production. Results of the 23 factorial experimental design showed that the highest dry biomass (0.66 g) was obtained under pH 6.0, inoculum concentration of 1 × 108 and 1% L-proline. However, the inoculum concentration was found to be statistically significant, the pH was marginally significant and the concentration of L-proline was not statistically significant. L-Asparaginase production varied between 0.58 and 1.02 U/g and did not reach the optimal point for enzyme production. This study demonstrates that T. catimbauensis is colonised by different Penicillium and Talaromyces species, which are indicated for enzyme production studies.


Assuntos
Asparaginase/biossíntese , Endófitos/enzimologia , Proteínas Fúngicas/biossíntese , Penicillium/enzimologia , Talaromyces/enzimologia , Tillandsia/microbiologia , Asparaginase/genética , Brasil , Endófitos/genética , Endófitos/isolamento & purificação , Florestas , Proteínas Fúngicas/genética , Penicillium/genética , Penicillium/isolamento & purificação , Filogenia , Talaromyces/genética , Talaromyces/isolamento & purificação
18.
World J Microbiol Biotechnol ; 34(9): 128, 2018 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-30083963

RESUMO

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.


Assuntos
Glucose Oxidase/biossíntese , Glucose Oxidase/genética , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Yarrowia/genética , Yarrowia/metabolismo , Sequência de Aminoácidos , Aspergillus niger/genética , Simulação por Computador , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose Oxidase/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/metabolismo , Software , Yarrowia/crescimento & desenvolvimento
19.
Appl Microbiol Biotechnol ; 102(20): 8621-8628, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30078136

RESUMO

The secretion of enzymes used by fungi to digest their environment has been exploited by humans for centuries for food and beverage production. More than a century after the first biotechnology patent, we know that the enzyme cocktails secreted by these amazing organisms have tremendous use across a number of industrial processes. Secreting the maximum titer of enzymes is critical to the economic feasibility of these processes. Traditional mutagenesis and screening approaches have generated the vast majority of strains used by industry for the production of enzymes. Until the emergence of economical next generation DNA sequencing platforms, the majority of the genes mutated in these screens remained uncharacterized at the sequence level. In addition, mutagenesis comes with a cost to an organism's fitness, making tractable rational strain design approaches an attractive alternative. As an alternative to traditional mutagenesis and screening, controlled manipulation of multiple genes involved in processes that impact the ability of a fungus to sense its environment, regulate transcription of enzyme-encoding genes, and efficiently secrete these proteins will allow for rational design of improved fungal protein production strains.


Assuntos
Proteínas Fúngicas/biossíntese , Fungos/metabolismo , Microbiologia Industrial , Proteínas Fúngicas/genética , Fungos/genética , Engenharia Metabólica
20.
Appl Microbiol Biotechnol ; 102(18): 7849-7863, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30032435

RESUMO

Laccase production and pellet formation of transformants of Coprinopsis cinerea strain FA2222 of C. cinerea laccase gene lcc1 subcloned behind the gpdII-promoter from Agaricus bisporus were compared with a control transformant carrying no extra laccase gene. At the optimum growth temperature of 37 °C, maximal laccase yields of 2.9 U/ml were obtained by the best lcc1 transformant pYSK7-26 in liquid shake flask cultures. Reduction in temperature to 25 °C increased laccase yields up to 9.2 U/ml. The control transformant had no laccase activities at 37 °C but native activity at 25 °C (3.5 U/ml). Changing the temperature had severe effects on the morphology of the mycelial pellets formed during cultivation, but links of distinct pellet morphologies to native or recombinant laccase production could not be established. Automated image analysis was used to characterise pellet formation and morphological parameters (pellet area, diameter, convexity and mycelial structure). Cross sections of selected pellets showed that they differentiated in an outer rind and an inner medulla of loosened hyphae. Pellets at 25 °C had a small and dense outer zone and adopted with time a smooth surface. Pellets at 37 °C had a broader outer zone and a fringy surface due to generation of more and larger protuberances in the rind that when released can serve for production of further pellets.


Assuntos
Agaricales/enzimologia , Agaricales/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Agaricales/genética , Técnicas de Cultura Celular por Lotes , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Lacase/genética , Micélio/enzimologia , Micélio/genética , Micélio/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Temperatura Ambiente
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