The current global scenario presents us with a growing increase in infections caused by fungi, referred to by specialists in the field as a "silent epidemic", aggravated by the limited pharmacological arsenal and increasing resistance to this therapy. For this reason, drug repositioning and therapeutic compound combinations are promising strategies to mitigate this serious problem. In this context, this study investigates the antifungal activity of the non-toxic, low-cost and widely available cationic polyelectrolyte Poly(diallyldimethylammonium chloride) (PDDA), in combination with different antifungal drugs: systemic (amphotericin B, AMB), topical (clioquinol, CLIO) and oral (nitroxoline, NTX). For each combination, different drug:PDDA ratios were tested and, through the broth microdilution technique, the minimum inhibitory concentration (MIC) of these drugs in the different ratios against clinically important Candida species strains was determined. Overall, PDDA combinations with the studied drugs demonstrated a significant increase in drug activity against most strains, reaching MIC reductions of up to 512 fold for the fluconazole resistant Candida krusei (Pichia kudriavzevii). In particular, the AMB-PDDA combination 1:99 was highly effective against AMB-resistant strains, demonstrating the excellent profile of PDDA as an adjuvant/association in novel antifungal formulations with outdated conventional drugs.
- MeSH
- amfotericin B farmakologie MeSH
- antifungální látky * farmakologie MeSH
- Candida * účinky léků MeSH
- fungální léková rezistence MeSH
- kandidóza mikrobiologie farmakoterapie MeSH
- kvartérní amoniové sloučeniny * farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti * MeSH
- Pichia MeSH
- polyelektrolyty farmakologie MeSH
- polyethyleny farmakologie chemie MeSH
- synergismus léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- amfotericin B MeSH
- antifungální látky * MeSH
- kvartérní amoniové sloučeniny * MeSH
- poly-N,N-dimethyl-N,N-diallylammonium chloride MeSH Prohlížeč
- polyelektrolyty MeSH
- polyethyleny MeSH
BACKGROUND AND OBJECTIVE: Yeasts have the remarkable capability to transform and integrate inorganic selenium into their cellular structures, thereby enhancing its bioavailability and reducing its toxicity. In recent years, yeasts have attracted attention as potential alternative sources of protein. METHODS: This study explores the selenium accumulation potential of two less explored yeast strains, namely the probiotic Saccharomyces boulardii CCDM 2020 and Pichia fermentas CCDM 2012, in comparison to the extensively studied Saccharomyces cerevisiae CCDM 272. Our investigation encompassed diverse stress conditions. Subsequently, the selenized yeasts were subjected to an INFOGEST gastrointestinal model. The adherence and hydrophobicity were determined with undigested cells RESULTS: Stress conditions had an important role in influencing the quantity and size of selenium nanoparticles (SeNPs) generated by the tested yeasts. Remarkably, SeMet synthesis was limited to Pichia fermentas CCDM 2012 and S. boulardii CCDM 2020, with S. cerevisiae CCDM 272 not displaying SeMet production at all. Throughout the simulated gastrointestinal digestion, the most substantial release of SeCys2, SeMet, and SeNPs from the selenized yeasts occurred during the intestinal phase. Notably, exception was found in strain CCDM 272, where the majority of particles were released during the oral phase. CONCLUSION: The utilization of both traditional and non-traditional selenized yeast types, harnessed for their noted functional attributes, holds potential for expanding the range of products available while enhancing their nutritional value and health benefits.
- Klíčová slova
- Antimicrobial activity, Cytotoxicity, INFOGEST, Selenium, Yeast,
- MeSH
- Pichia MeSH
- probiotika * metabolismus MeSH
- Saccharomyces boulardii * metabolismus MeSH
- Saccharomyces cerevisiae chemie MeSH
- selen * metabolismus MeSH
- trávení MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- selen * MeSH
Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for β-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for β-glucosidase production. The results showed that at 72 h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55 g/L, respectively, while the total mutant β-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60 h of cultivation, the highest total mutant β-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant β-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73 g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 gcell/gMeOH, methanol consumption rate ([Formula: see text]) 5.76 gMeOH/h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and β- glucosidase activity were produced under exponential feed rate than constant and linear feed rates.
- Klíčová slova
- Exponential feed rate, Fed-batch fermentation, Methanol feeding profile, Pichia pastoris, β-glucosidase,
- MeSH
- bioreaktory MeSH
- celulasy * metabolismus MeSH
- fermentace MeSH
- methanol * metabolismus MeSH
- Pichia metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- celulasy * MeSH
- methanol * MeSH
- rekombinantní proteiny MeSH
Systemic infections caused by pathogenic Candida species pose a significant threat to public health in the past decades due to increasing resistance to existing antifungal drugs. Given this scenario, probiotics have been suggested as an alternative approach for managing Candida infections. Hence, the purpose of this study was to evaluate whether probiotic yeast Saccharomyces boulardii co-aggregate with Candida spp. as well as to determine their auto-aggregation ability in dependence on temperature (28 °C, 37 °C, 42 °C) and pH (4.5, 7.0, 8.5) after 5 h and 24 h. Our results revealed that the aggregation of tested yeasts was lower in the first 5 h but increased significantly after 24 h. All strains were able to auto-aggregate in different degrees ranging from 47.46 to 95.95% assessed at 24 h of incubation. Among them the highest auto-aggregation values had C. albicans and C. krusei strains followed by probiotic strain S. boulardii, while the less were observed in C. glabrata strains. In addition, co-aggregation between probiotic and Candida strains was strain-specific. It was evident that S. boulardii significantly inhibited the aggregation of C. albicans ATCC 10261, C. krusei ATCC 6258, and C. glabrata ZIM 2369. However, in C. glabrata ZIM 2382, the aggregation was even enhanced. Temperature and pH also affected the ability to aggregate in a different way only after 5 h of incubation, with the highest cell aggregation evidenced at temperature 37 °C in most cases and pH 4.5. These findings may be of importance when trying to establish probiotic use against pathogenic Candida species.
Fructooligosaccharides (FOS) are compounds possessing various health properties and are added to functional foods as prebiotics. The commercial production of FOS is done through the enzymatic transfructolysation of sucrose by β-fructofuranosidases which is found in various organisms of which Aureobasidium pullulans and Aspergillus niger are the most well known. This study overexpressed two differently codon-optimized variations of the Aspergillus fijiensis β-fructofuranosidase-encoding gene (fopA) under the transcriptional control of either the alcohol oxidase (AOX1) or glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters. When cultivated in shake flasks, the two codon-optimized variants displayed similar volumetric enzyme activities when expressed under control of the same promoter with the GAP strains producing 11.7 U/ml and 12.7 U/ml, respectively, and the AOX1 strains 95.8 U/ml and 98.6 U/ml, respectively. However, the highest production levels were achieved for both codon-optimized genes when expressed under control of the AOX1 promoter. The AOX1 promoter was superior to the GAP promoter in bioreactor cultivations for both codon-optimized genes with 13,702 U/ml and 2718 U/ml for the AOX1 promoter for ATUM and GeneArt®, respectively, and 6057 U/ml and 1790 U/ml for the GAP promoter for ATUM and GeneArt®, respectively. The ATUM-optimized gene produced higher enzyme activities when compared to the one from GeneArt®, under the control of both promoters.
- MeSH
- Aspergillus MeSH
- invertasa * genetika MeSH
- kodon genetika MeSH
- Pichia * genetika MeSH
- rekombinantní proteiny genetika MeSH
- Saccharomycetales MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- invertasa * MeSH
- kodon MeSH
- rekombinantní proteiny MeSH
This study was designed to evaluate the prevalence of antifungal resistance, genetic mechanisms associated with in vitro induction of azole and echinocandin resistance and genotyping of Candida krusei, which is intrinsically resistant to fluconazole and is recovered from clinical and nonclinical sources from different countries. Our results indicated that all the isolates were susceptible or had the wild phenotype (WT) to azoles, amphotericin B, and only 1.27% showed non-WT for flucytosine. Although 70.88% of the isolates were resistant to caspofungin, none of them were categorized as echinocandin-resistant as all were susceptible to micafungin and no FKS1 hot spot 1 (HS1) or HS2 mutations were detected. In vitro induction of azole and echinocandin resistance confirmed the rapid development of resistance at low concentrations of fluconazole (4 μg/ml), voriconazole (0.06 μg/ml), and micafungin (0.03 μg/ml), with no difference between clinical and nonclinical isolates in the resistance development. Overexpression of ABC1 gene and FKS1 HS1 mutations were the major mechanisms responsible for azole and echinocandin resistance, respectively. Genotyping of our 79 isolates coupled with 217 other isolates from different sources and geography confirmed that the isolates belong to two main subpopulations, with isolates from human clinical material and Asia being more predominant in cluster 1, and environmental and animals isolates and those from Europe in cluster 2. Our results are of critical concern, since realizing that the C. krusei resistance mechanisms and their genotyping are crucial for guiding specific therapy and for exploring the potential infection source.
- Klíčová slova
- C. krusei, Candida genotyping, azole resistance, echinocandin resistance, human-animal transmission,
- MeSH
- antifungální látky farmakologie terapeutické užití MeSH
- azoly * farmakologie MeSH
- echinokandiny * farmakologie MeSH
- fungální léková rezistence genetika MeSH
- genotyp MeSH
- mikrobiální testy citlivosti MeSH
- Pichia MeSH
- prevalence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antifungální látky MeSH
- azoly * MeSH
- echinokandiny * MeSH
Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5 mg/L ciprofloxacin (CIP) and 99% of 100 mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.
- MeSH
- antibakteriální látky farmakologie MeSH
- lakasa * genetika metabolismus MeSH
- Pichia * genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- rekombinantní proteiny genetika MeSH
- Saccharomycetales MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- lakasa * MeSH
- rekombinantní proteiny MeSH
Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell's physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia's potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. KEY POINTS: • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.
- Klíčová slova
- Chaperone, Co-expression strategy, Folding and secretion, Pichia pastoris, Productivity of recombinant protein production, Unfolded protein response (UPR),
- MeSH
- fungální proteiny * genetika metabolismus MeSH
- Pichia * genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- Saccharomycetales MeSH
- signální dráha UPR MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- fungální proteiny * MeSH
- rekombinantní proteiny MeSH
The β-N-acetylhexosaminidase from Penicillium oxalicum (PoHex; EC 3.2.1.52) is a fungal glycosidase with an outstandingly high GalNAcase/GlcNAcase activity ratio. It has a remarkable synthetic capability and can process carbohydrates functionalized at various positions. However, the production in the native fungal host is lengthy, unselective and purification from the fungal medium is complicated and low yielding. We present here a novel production method of this enzyme in the eukaryotic host of Pichia pastoris, followed by elegant one-step purification to homogeneity. The resulting recombinant enzyme has improved biochemical and catalytic properties compared to the fungal wild type. Its good production yield (11 mg/400 mL cultivation medium) greatly expands the scope of synthetic applications. We further demonstrate the synthetic utility and broad acceptor specificity of recombinant PoHex in the glycosylation of a series of challenging acceptors with varying structural architectures, namely secondary and tertiary hydroxyl, aldoxime and a poly-hydroxylated compound.
- Klíčová slova
- Glycosylation, Penicillium oxalicum, Pichia pastoris, Substrate specificity, β-N-acetylhexosaminidase,
- MeSH
- beta-N-acetylhexosaminidasy genetika metabolismus MeSH
- glykosylace MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- Penicillium enzymologie MeSH
- Pichia metabolismus MeSH
- rekombinantní proteiny biosyntéza izolace a purifikace metabolismus MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- beta-N-acetylhexosaminidasy MeSH
- rekombinantní proteiny MeSH
AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-β-glucosidase I and II (αRβG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-β-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRβG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRβG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRβG I. On the other hand, αRβG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRβG I showed exclusive specificity toward 7-O-β-rutinosyl flavonoids, whereas αRβG II hydrolyzed both 7-O-β-rutinosyl- and 3-O-β-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-β-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRβG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.
- Klíčová slova
- Enzyme catalysis, Glycoside hydrolase, Hesperidin, Recombinant protein, Rutin,
- MeSH
- Acremonium enzymologie genetika MeSH
- flavonoidy metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- fylogeneze MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- molekulová hmotnost MeSH
- Pichia genetika MeSH
- rekombinantní proteiny metabolismus MeSH
- sekvenční analýza DNA MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- flavonoidy MeSH
- fungální proteiny MeSH
- glykosidhydrolasy MeSH
- rekombinantní proteiny MeSH
