Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.495
Filtrar
1.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608301

RESUMO

Volatile organic compounds (VOCs) are chemicals responsible for antagonistic activity between microorganisms. The impact of VOCs on microbial community succession of fermentation is not well understood. In this study, Pichia spp. were evaluated for VOC production as a part of antifungal activity during baijiu fermentation. The results showed that the abundance of Pichia in the defect group (agglomerated fermented grains) was lower than that in control group, and a negative interaction between Pichia and Monascus was determined (P < 0.05). In addition, the disruption of fungi was significantly related to the differences of metabolic profiles in fermented grains. To determine production of VOCs from Pichia and its effect on Monascus purpureus, a double-dish system was assessed, and the incidence of M. purpureus reduction was 39.22% after 7 days. As to antifungal volatile compounds, 2-phenylethanol was identified to have an antifungal effect on M. purpureus through contact and noncontact. To further confirm the antifungal activity of 2-phenylethanol, scanning electron microscopy showed that 2-phenylethanol widely and significantly inhibited conidium germination and mycelial growth of filamentous fungi. Metatranscriptomic analysis revealed that the Ehrlich pathway is the metabolic path of 2-phenylethanol in Pichia and identified potential antifungal mechanisms, including protein synthesis and DNA damage. This study demonstrated the role of volatile compound-mediated microbial interaction in microbiome assembly and discovered a plausible scenario in which Pichia antagonized fungal blooms. The results may improve the niche establishment and growth of the functional yeast that enhances the flavor of baijiu.IMPORTANCE Fermentation of food occurs within communities of interacting species. The importance of microbial interactions in shaping microbial structure and metabolic performance to optimize the traditional fermentation process has long been emphasized, but the interaction mechanisms remain unclear. This study applied metabolome analysis and amplicon sequencing along with metatranscriptomic analysis to examine the volatile organic compound-mediated antifungal activity of Pichia and its effect on the metabolism of ethanol during baijiu fermentation, potentially enhancing the establishment of the fermentation niche and improving ethanol metabolism.


Assuntos
Agentes de Controle Biológico/metabolismo , Etanol/metabolismo , Monascus/crescimento & desenvolvimento , Pichia/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Bebidas Alcoólicas , Bactérias/metabolismo , Dano ao DNA , DNA Fúngico , Fermentação , Metaboloma , Monascus/genética , Biossíntese de Proteínas
2.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530422

RESUMO

The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non-Saccharomyces yeasts such as Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, and Pichia kluyveri have become popular with regard to winemaking and improved wine quality. For that reason, several manufacturers started to offer commercially available strains of these non-Saccharomyces species. P. kluyveri stands out, mainly due to its contribution to wine aroma, glycerol, ethanol yield, and killer factor. The metabolism of the yeast allows it to increase volatile molecules such as esters and varietal thiols (aroma-active compounds), which increase the quality of specific varietal wines or neutral ones. It is considered a low- or non-fermentative yeast, so subsequent inoculation of a more fermentative yeast such as Saccharomyces cerevisiae is indispensable to achieve a proper fermented alcohol. The impact of P. kluyveri is not limited to the grape wine industry; it has also been successfully employed in beer, cider, durian, and tequila fermentation, among others, acting as a promising tool in those fermentation processes. Although no Pichia species other than P. kluyveri is available in the regular market, several recent scientific studies show interesting improvements in some wine quality parameters such as aroma, polysaccharides, acid management, and color stability. This could motivate yeast manufacturers to develop products based on those species in the near future.


Assuntos
Bioengenharia , Fermentação , Microbiologia Industrial , Pichia/metabolismo , Vinho , Bioengenharia/métodos , Ecologia , Qualidade dos Alimentos , Pichia/classificação , Vitis/química , Vitis/microbiologia , Vinho/análise , Vinho/normas
3.
Food Chem ; 346: 128899, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33401089

RESUMO

It is now well established that ß-glucosidases (BGLs) from non-Saccharomyces yeasts are key enzymes that hydrolyze grape-derived aroma precursors enhancing the flavour of wines. This work reports on the specificity for wine glycosides and the impact on wine aroma, of three native yeast ß-glucosidases. Volatile compounds were analyzed by gas-chromatography and mass spectroscopy (GC-MS) and wine aroma was studied by sensory analysis. Issatchenkia terricola ß-glucosidase stood out from the other ß-glucosidases studied. The I. terricola BGL showed remarkable specificity for norisoprenoid aglycones such as: 3-oxo-7, 8-dihydro-alpha-ionol, 3-oxo-α-ionol, vomifoliol. This different specificity was perceived in the sensory tests. The judges described pleasant fruity, sweet, honey and raisin notes in both Tannat and Muscat wines treated with I. terricola BGL. These results are particularly remarkable for Tannat wines, since there are few reports concerning the application of ß-glucosidases to enhance its aroma of Tannat, and none with BGLs from native yeasts.


Assuntos
Odorantes/análise , Vitis/química , Vinho/análise , beta-Glucosidase/metabolismo , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Paladar , Vitis/microbiologia
4.
Food Chem ; 346: 128897, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33406455

RESUMO

Pichia fermentans Z9Y-3 and its intracellular enzymes were inoculated along with S. cerevisiae in synthetic grape must to modulate fruity ester production. The levels of ester-related enzymes, ester precursors, and fruity esters were monitored every 24 h during fermentation. Results showed that the levels of ethyl acetate, acetate higher alcohol esters (AHEs), short chain fatty acid ethyl esters (SFEs), and medium chain fatty acid ethyl esters (MFEs) were significantly enhanced in mixed fermentation. Pearson correlation analysis further revealed that higher alcohols and fatty acids played a more important role in fruity ester production than enzymes; Particularly, the correlation coefficient between fatty acids and MFEs was 0.940. In addition, supplementation of medium chain fatty acids (7.2 mg/L) at the metaphase of single S. cerevisiae fermentation improved ethyl acetate, AHE, SFE, and MFE production by 42.56%, 21.00%, 61.33%, and 90.04%, respectively, although the high level of ethyl acetate might result in off-flavors.


Assuntos
Ésteres/química , Ésteres/metabolismo , Ácidos Graxos/metabolismo , Fermentação , Frutas/química , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Paladar , Vitis/química , Vitis/microbiologia , Vinho/análise
5.
Food Chem ; 346: 128953, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33412487

RESUMO

Tartary buckwheat is widely accepted as its nutritionalvalue. Some allergic reactions hinder its utilization. This research focused on evaluating the core epitope of 16 kDa allergen (Fag t 2) in tartary buckwheat. Six B- and seven T cell epitopes of Fag t 2 were predicted, and six B cell epitope-mutants were expressed in Pichia pastoris. Bioinformatics analysis and SDS-PAGE demonstrated that the molecular weight, isoelectric point and spatial structures of six mutant allergens were similar with Fag t 2, with the same signal peptide sequences and α-amylase inhibitor domain. There was no significant change in mutants' spatial conformation confirmed by Circular Dichroism. The position K132N and peptides at 108-117 and 132-141 were the core B- and T cell epitopes of Fag t 2 confirmed by competitive inhibition ELISA and dot blot. This result was of great significance on the study of allergen epitopes in prevention and treatment of hypersensitivity.


Assuntos
Alérgenos/imunologia , Epitopos/imunologia , Fagopyrum/metabolismo , Alérgenos/química , Alérgenos/genética , Alérgenos/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/metabolismo , Mutagênese Sítio-Dirigida , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
6.
Food Microbiol ; 94: 103629, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33279061

RESUMO

De novo sourdough cultures were here assessed for their potential as sources of yeast strains for low-alcohol beer brewing. NGS analysis revealed an abundance of ascomycete yeasts, with some influence of grain type on fungal community composition. Ten different ascomycete yeast species were isolated from different sourdough types (including wheat, rye, and barley) and seven of these were screened for a number of brewing-relevant phenotypes. All seven were maltose-negative and produced less than 1% (v/v) alcohol from a 12 °Plato wort in initial fermentation trials. Strains were further screened for their bioflavouring potential (production of volatile aromas and phenolic notes, reduction of wort aldehydes), stress tolerance (temperature extremes, osmotic stress and ethanol tolerance) and flocculence. Based on these criteria, two species (Kazachstania servazzii and Pichia fermentans) were selected for 10 L-scale fermentation trials and sensory analysis of beers. The latter species was considered particularly suitable for production of low-alcohol wheat beers due to its production of the spice/clove aroma 4-vinylguaiacol, while the former showed potential for lager-style beers due to its clean flavour profile and tolerance to low temperature conditions.


Assuntos
Álcoois/análise , Cerveja/microbiologia , Pão/microbiologia , Maltose/metabolismo , Pichia/metabolismo , Saccharomycetales/metabolismo , Álcoois/metabolismo , Cerveja/análise , Fermentação , Aromatizantes/análise , Aromatizantes/metabolismo , Hordeum/metabolismo , Hordeum/microbiologia , Odorantes , Secale/metabolismo , Secale/microbiologia , Triticum/metabolismo , Triticum/microbiologia
7.
Int J Food Microbiol ; 337: 108933, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33181418

RESUMO

Chinese Baijiu is prepared using multiple microbial strains and complex metabolites by simultaneous saccharification and fermentation (SSF). Yeasts are challenged by various endogenous and exogenous factors, detrimentally affecting the ethanol yield. It is imperative to identify and control inhibitory factors. In the present study, microbial taxa and metabolites during Baijiu fermentation were evaluated to identify inhibitors of ethanol production. We found that filamentous fungi and Bacillus, contributing to saccharification, were negatively related to the ethanol content (Spearman's |ρ| > 0.5, P < 0.05). To explore how they affect ethanol production, ten filamentous fungi and three Bacillus strains were isolated. In addition to glucose and maltose, polyols were simultaneously generated by filamentous fungi and Bacillus via the hydrolysis of starch, among which mannitol and erythritol had the highest contents of up to 41.56 ± 2.01 g/kg and 16.16 ± 1.13 g/kg, respectively. The presence of mannitol and erythritol inhibited ethanol production by the functional yeasts Saccharomyces cerevisiae and Pichia kudriavzevii. The presence of 10.0 g/L mannitol significantly (P < 0.01) decreased the ethanol yield of S. cerevisiae by 12.67% (from 39.34 ± 0.02% to 32.71 ± 0.49%). These results revealed that polyols may inhibit the production of Baijiu and other fermented foods, suggesting that the origin and influence of polyols should be a focus of future research.


Assuntos
Eritritol/metabolismo , Etanol/metabolismo , Fermentação , Alimentos e Bebidas Fermentados/microbiologia , Manitol/metabolismo , Humanos , Hidrólise , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo
8.
Int J Food Microbiol ; 339: 109015, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33340944

RESUMO

Glucose and fructose are the main fermentable sugars in cocoa pulp. During fermentation, glucose is consumed within 48-72 h and fructose only after 120 h, mainly associated with the preferential use of glucose by microorganisms. In the first stage of this study, the complete genome sequence of a lactic acid bacterium with high fructose consumption capacity (Lactobacillus plantarum LPBF35) was reported. The notable genomic features of L. plantarum LPBF35 were the presence of alcohol/acetaldehyde dehydrogenase gene and improved PTS system, confirming its classification as a "facultatively" fructophilic bacterium. Subsequently, this bacterium was introduced into cocoa fermentation process in single and mixed cultures with Pediococcus acidilactici LPBF66 or Pichia fermentans YC5.2. Community composition by Illumina-based amplicon sequencing and viable counts indicated suppression of wild microflora in all treatments. At the beginning of the fermentation processes, cocoa pulp consisted of approximately 73.09 mg/g glucose and 73.64 mg/g fructose. The L. plantarum LPBF35 + P. fermentans YC5.2 process showed the lowest levels of residual sugars after 72 h of fermentation (7.89 and 4.23 mg/g, for fructose and glucose, respectively), followed by L. plantarum LPBF35 + Ped. acidilactici LPBF66 (8.85 and 6.42 mg/g, for fructose and glucose, respectively), single L. plantarum LPBF35 treatment (4.15 and 10.15 mg/g, for fructose and glucose, respectively), and spontaneous process (22.25 and 14.60 mg/g, for fructose and glucose, respectively). The positive interaction between L. plantarum LPBF35 and P. fermentans YC5.2 resulted in an improved formation of primary (ethanol, lactic acid, and acetic acid) and secondary (2-methyl-1-butanol, isoamyl acetate, and ethyl acetate) metabolites during fermentation. The primary metabolites accumulated significantly in cocoa beans fermented by P. fermentans YC5.2 + L. plantarum LPBF35, causing important reactions of color development and key flavor molecules formation. The results of this study suggest that fructophilic lactic acid bacteria and yeast is a microbial consortium that could improve sugar metabolism and aroma formation during cocoa beans fermentation.


Assuntos
Cacau/metabolismo , Cacau/microbiologia , Fermentação , Microbiologia de Alimentos , Lactobacillus plantarum/metabolismo , Interações Microbianas , Açúcares/metabolismo , Ácido Acético/metabolismo , Lactobacillus plantarum/genética , Lactobacillus plantarum/crescimento & desenvolvimento , Odorantes , Pediococcus acidilactici/metabolismo , Pichia/metabolismo
9.
Int J Food Microbiol ; 333: 108796, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-32771820

RESUMO

The objective of this study was to investigate the role of yeasts in the wet fermentation of coffee beans and their contribution to coffee quality using a novel approach. Natamycin (300 ppm) was added to the fermentation mass to suppress yeast growth and their metabolic activities, and the resultant microbial ecology, bean chemistry and sensory quality were analyzed and compared to non-treated spontaneous fermentation we reported previously. The yeast community was dominated by Hanseniaspora uvarum and Pichia kudriavzevii and grew to a maximum population of about 5.5 log CFU/g in the absence of Natamycin, while when Natamycin was added yeasts were suppressed. The major bacterial species in both the spontaneous and yeast-suppressed fermentations included the lactic acid bacteria Leuconostoc mesenteroides and Lactococcus lactis, the acetic acid bacteria Gluconobacter cerinus and Acetobacter persici and the Enterobacteriaceae Enterobacter, Citrobacter and Erwinia. For both fermentations, the mucilage layers were completely degraded by the end of the process and the absence of yeast activities had no significant impact on mucilage degradation. During fermentation, reducing sugars were consumed while lactic acid was accumulated inside the beans, and its concentration was significantly higher in the spontaneous fermentation (3 times) than that where yeasts were suppressed by Natamycin. Glycerol was detected with a concentration of 0.08% in the absence of Natamycin and was not identified when Natamycin was added. Green beans fermented with yeast growth contained a higher amount of isoamyl alcohol (21 times), ethanol (3.7 times), acetaldehyde (8 times), and ethyl acetate (25 times) compared to beans fermented in the absence of yeast activities, which remained higher in the former after roasting. Beans fermented without yeast activities had a mild fruity aroma, and lower sensory scores of fragrances (7.0), flavor (6.5), acidity (6.3), body (7.0) and overall score (6.5) compared to the former. These findings demonstrated the crucial roles of yeasts in wet fermentation of coffee beans and for producing high quality coffee.


Assuntos
Bactérias/metabolismo , Café/metabolismo , Fermentação/fisiologia , Hanseniaspora/metabolismo , Pichia/metabolismo , Leveduras/metabolismo , Acetaldeído/metabolismo , Acetatos/metabolismo , Ácido Acético/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/classificação , Reatores Biológicos/microbiologia , Café/microbiologia , Etanol/metabolismo , Ácido Láctico/metabolismo , Natamicina/farmacologia , Odorantes/análise , Pentanóis/metabolismo , Paladar
10.
Life Sci ; 260: 118274, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827545

RESUMO

AIMS: Triple-negative breast cancer (TNBC) is not sensitive to current endocrine treatments, so new treatment strategies need to be explored. Based on previous antitumour studies on anti-TNFα nanobody, we designed a novel fusion nanobody to enhance antitumour activity of the anti-TNFα nanobody in TNBC. MAIN METHODS: The RGD4C contains RGD sequence, which is the smallest recognition unit binding to the αvß3 receptor on tumour cell membranes and involved in tumour cell adhesion, proliferation, and metastasis. RGD4C was fused to anti-TNFα nanobody to investigate the antitumour activity in vitro and in vivo. KEY FINDINGS: The antitumour effects of fusion nanobody V-L-R-H could effectively bind to αvß3 and inhibit cell migration and proliferation of MDA-MB-231, which had satisfying purification efficiency and approving antigen or receptor binding activity. V-L-R-H could inhibit the TNFα-mediated PI3K/AKT/NF-κB signal pathway and integrin αvß3 correlative FAK focal adhesion signal pathway. Mouse xenograft tumour experiments showed that the V-L-R-H could inhibit tumour proliferation and metastasis; reduce the TNFα, HIFα, Ki67, and CD31 concentrations in tumour; and inhibit the process of epithelial-mesenchymal transition. SIGNIFICANCE: The fusion nanobody enhanced antitumour activity of the anti-TNFα nanobody on TNBC. It provided a reference for the design of dual functional fusion proteins and development of tumour treatment strategies of antagonistic TNFα and αvß3, and a new therapeutic strategy and research direction for the treatment of TNBC.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Domínio Único/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Fator de Necrose Tumoral alfa/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Expressão Gênica , Humanos , Integrina alfaVbeta3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/prevenção & controle , Oligopeptídeos/química , Oligopeptídeos/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Anticorpos de Domínio Único/genética , Neoplasias de Mama Triplo Negativas/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Food Chem ; 332: 127426, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32619948

RESUMO

A selected Pichia fermentans strain was simultaneously and sequentially inoculated in synthetic and real juice with S. cerevisiae strains of different antagonistic activities in a ratio 1:1 to observe the correlation between varietal odorants and glycosidase activities. Fermentations using pure S. cerevisiae strains were used for comparison. Yeast biomass and glycosidase activities were monitored, varietal odorants were detected using HS-SPME-GC/MS during fermentation. The final wine aroma attributes were analyzed by trained panelists. Results showed that co-inoculation with high antagonistic S. cerevisiae resulted in higher glycosidase activities than others. Pearson correlation analysis indicated that yeast biomass was positively related to glycosidase activities during fermentation. The increase in glycosidase activities was the main reason for the higher production of terpenes and C13-norisoprenoids, and for the lower C6 compound content, which lead to superior fruity and floral aromas in the final wine samples of the high antagonistic S. cerevisiae group.


Assuntos
Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/análise , Fermentação , Frutas/química , Sucos de Frutas e Vegetais/análise , Sucos de Frutas e Vegetais/microbiologia , Cromatografia Gasosa-Espectrometria de Massas , Norisoprenoides/análise , Norisoprenoides/metabolismo , Odorantes/análise , Pichia/enzimologia , Pichia/crescimento & desenvolvimento , Terpenos/análise , Terpenos/metabolismo
12.
Food Chem ; 333: 127527, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32683263

RESUMO

The aim of this study was to develop a novel fermented soybean food (FSF) using selected Bacillus subtilis GD1, Bacillus subtilis N4, Bacillus velezensis GZ1, Lactobacillus delbrueckii subsp. bulgaricus and Hansenula anomala, as well as to assess its antioxidant and anti-fatigue activity. These Bacillus strains had excellent enzyme producing and soybean transformation capacity. FSF showed the highest peptide, total phenol, total flavonoid content, antioxidant activity, and suitable organic acid and biological amine content. In intense exercise mice, FSF treatment markedly increased hepatic glycogen level, decreased metabolite accumulation, improved the activities of antioxidant enzymes and decreased malondialdehyde (MDA) level in serum and liver, respectively. Furthermore, FSF treatment increased nuclear factor-erythroid 2-related factor 2 (Nrf2) and antioxidant response element (ARE)-dependent gene expression. Together, the selection of microbial starter culture and mixed culture fermentation are essential for the effective enrichment of bioactive compounds, and FSF has stronger antioxidant and anti-fatigue activity.


Assuntos
Antioxidantes/metabolismo , Bacillus/metabolismo , Alimentos e Bebidas Fermentados/análise , Lactobacillus/metabolismo , Pichia/metabolismo , Soja/metabolismo , Animais , Antioxidantes/farmacologia , Bacillus/crescimento & desenvolvimento , Nitrogênio da Ureia Sanguínea , Fadiga/metabolismo , Fadiga/patologia , Flavonoides/análise , Glicogênio/metabolismo , Lactobacillus/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Malondialdeído/sangue , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/análise , Condicionamento Físico Animal , Pichia/crescimento & desenvolvimento , Soja/química
13.
Mol Genet Genomics ; 295(5): 1295-1304, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32566991

RESUMO

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the - 973 base pair (bp) to - 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5'-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.


Assuntos
Metanol/metabolismo , Pichia/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aldeído Oxidase/metabolismo , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Deleção de Sequência , Fatores de Transcrição/química , Sítio de Iniciação de Transcrição
14.
Proc Natl Acad Sci U S A ; 117(27): 16043-16054, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571919

RESUMO

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.


Assuntos
Antifúngicos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Simbiose , Sequência de Aminoácidos , Botrytis/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Cisteína/química , Fusarium/metabolismo , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/microbiologia , Espectroscopia de Ressonância Magnética , Medicago truncatula/microbiologia , Pichia/metabolismo , Doenças das Plantas/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia
15.
Food Chem ; 328: 127110, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32464557

RESUMO

In the present work we aimed to demonstrate the influence of inoculum starter in support high quality fermentation. Cocoa fermentations were performed in wooden boxes and eight yeasts strains were used in separated fermentations of fine cocoa, type Scavina, as starter inoculum. Temperature, pH, titirable acidity, reducing sugar and free amino acids were evaluated during or after fermentation. The influence of starters yeasts on the decrease of acidity, sugar concentration and free amino acids was significant. The strains Candida parapsilosis, Torulaspora delbrueckii and Pichia kluyveri showed greater changes in the reducing sugar and free amino acids in fermented cocoa beans. These results indicate the ability of yeast used as inoculum starter to modify the end condition and further enhance the quality of fine cocoa beans.


Assuntos
Cacau , Microbiologia de Alimentos/métodos , Leveduras , Aminoácidos/análise , Aminoácidos/metabolismo , Cacau/química , Cacau/metabolismo , Candida parapsilosis/genética , Candida parapsilosis/metabolismo , Chocolate , Fermentação , Concentração de Íons de Hidrogênio , Pichia/genética , Pichia/metabolismo , Sementes/química , Sementes/microbiologia , Temperatura , Torulaspora/genética , Torulaspora/metabolismo , Leveduras/genética , Leveduras/metabolismo
16.
Appl Environ Microbiol ; 86(15)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32471916

RESUMO

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Glucose/metabolismo , Saccharomycetales/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Oxirredução , Pichia/enzimologia , Pichia/metabolismo , Saccharomycetales/enzimologia
17.
Enzyme Microb Technol ; 137: 109552, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423672

RESUMO

Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD+ via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD+). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications.


Assuntos
Chaetomium/enzimologia , Formiato Desidrogenases/biossíntese , Pichia/metabolismo , Aminoácidos/química , Catálise , Chaetomium/genética , Meios de Cultura/química , Fermentação , Oxirredução , Pichia/genética , Engenharia de Proteínas
18.
Anal Bioanal Chem ; 412(10): 2365-2374, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32130438

RESUMO

In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level. Graphical Abstract.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Lipidômica/métodos , Lipídeos/química , Espectrometria de Massas/métodos , Humanos , Metabolismo dos Lipídeos , Lipídeos/sangue , Pichia/química , Pichia/metabolismo , Plasma/química
19.
Arch Virol ; 165(5): 1057-1067, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144542

RESUMO

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.


Assuntos
Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Infecções por Vírus Respiratório Sincicial/patologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/isolamento & purificação , Células Th1/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas Virais de Fusão/genética , Viremia/imunologia
20.
J Biosci Bioeng ; 130(1): 29-35, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32171656

RESUMO

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.


Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pichia/enzimologia , Torulaspora/enzimologia , Clonagem Molecular , D-Xilulose Redutase/química , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Cinética , Pichia/genética , Pichia/metabolismo , Torulaspora/genética , Torulaspora/metabolismo , Xilitol/metabolismo , Xilose/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...