Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis.

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

BACKGROUND: Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. OBJECTIVE: The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. METHODS: Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, in vitro experiments on fitness cost and virulence were carried out. RESULTS: Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. CONCLUSION: In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

Using Extracted Sugars from Spoiled Date Fruits as a Sustainable Feedstock for Ethanol Production by New Yeast Isolates.

This study focuses on investigating sugar recovery from spoiled date fruits (SDF) for sustainable ethanol production using newly isolated yeasts. Upon their isolation from different food products, yeast strains were identified through PCR amplification of the D1/D2 region and subsequent comparison with the GenBank database, confirming isolates KKU30, KKU32, and KKU33 as Saccharomyces cerevisiae; KKU21 as Zygosaccharomyces rouxii; and KKU35m as Meyerozyma guilliermondii. Optimization of sugar extraction from SDF pulp employed response surface methodology (RSM), varying solid loading (20-40%), temperature (20-40 °C), and extraction time (10-30 min). Linear models for sugar concentration (R1) and extraction efficiency (R2) showed relatively high R2 values, indicating a good model fit. Statistical analysis revealed significant effects of temperature and extraction time on extraction efficiency. The results of batch ethanol production from SDF extracts using mono-cultures indicated varying consumption rates of sugars, biomass production, and ethanol yields among strains. Notably, S. cerevisiae strains exhibited rapid sugar consumption and high ethanol productivity, outperforming Z. rouxii and M. guilliermondii, and they were selected for scaling up the process at fed-batch mode in a co-culture. Co-cultivation resulted in complete sugar consumption and higher ethanol yields compared to mono-cultures, whereas the ethanol titer reached 46.8 ± 0.2 g/L.

Optimizing the synthesis of yeast Beta-glucan via response surface methodology for nanotechnology application.

BACKGROUND: The production of biopolymers from waste resources is a growing trend, especially in high-population countries like Egypt. Beta-glucan (ß-glucan) belongs to natural polysaccharides that are derived from plant and microbial origins. In this study, following increasing demands for ß-glucan owing to its bioactive properties, a statistical model to enhance microbial ß-glucan production was evaluated for its usefulness to the food and pharmaceutical industries. In addition, a trial to convert ß-glucan polymer to nanostructure form was done to increase its bioactivity. RESULTS: Ingredients of low-cost media based on agro-industrial wastes were described using Plackett-Burman and central composite design of response surface methodology for optimizing yeast ß-glucan. Minerals and vitamin concentrations significantly influenced ß-glucan yield for Kluyveromyces lactis and nitrogen and phosphate sources for Meyerozyma guilliermondii. The maximum predicted yields of ß-glucan recovered from K. lactis and M. guilliermondii after optimizing the medium ingredients were 407 and 1188 mg/100 ml; respectively. For the first time, yeast ß-glucan nanoparticles (ßGN) were synthesized from the ß-glucan polymer using N-dimethylformamide as a stabilizer and characterized using UV-vis spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The average size of ßGN was about 300 nm as determined by DLS. The quantitative variation of functional groups between ß-glucan polymer and ßGN was evaluated by FT-IR for explaining the difference in their biological activity against Normal Homo sapiens-Hela contaminant and Hepatic cancer cell lines. CONCLUSIONS: Enriching the low-cost media based on agro-industrial wastes with nutritional ingredients improves the yield of yeast ß-glucan. The present study succeeds to form ß-glucan nanoparticles by a simple method.

Enolase in Meyerozyma guilliermondii strain SO: Sequential and structural insights of MgEno4581 as a putative virulence factor and host-fungal interactions through comprehensive in silico approaches.

Meyerozyma guilliermondii is a rare opportunistic fungal pathogen that causes deadly invasive candidiasis in human. M. guilliermondii strain SO is a local yeast isolate that possesses huge industrial interests but also pathogenic towards zebrafish embryos. Enolases that bind to human extracellular matrix (ECM) proteins are among the fungal virulence factors. To understand its pathogenicity mechanism down to molecular level, especially in the rare M. guilliermondii, this study aimed to identify and characterize the potentially virulence-associated enolase in M. guilliermondii strain SO using bioinformatics approaches. Profile Hidden-Markov model was implemented to identify enolase-related sequences in the fungal proteome. Sequence analysis deciphered only one (MgEno4581) out of nine sequences exhibited potent virulence traits observed similarly in the pathogenic Candida albicans. MgEno4581 structure that was predicted via SWISS-MODEL using C. albicans enolase (CaEno1; PDB ID: 7vrd) as the homology modeling template portrayed a highly identical motif with CaEno1 that facilitates ECM proteins binding. Amino acid substitutions (D234K, K235A, Y238H, K239D, G243K, V248C and Y254F) in ECM-binding motif of Saccharomyces cerevisiae enolase (ScEno) compared to MgEno4581 and CaEno1 caused changes in motif's surface charges. Protein-protein docking indicated F253 in ScEno only interacted hydrophobically with human plasminogen (HPG). Hydrogen linkages were observed for both MgEno4581 and CaEno1, suggesting a stronger interaction with HPG in the hydrophilic host microenvironments. Thus, our in silico characterizations on MgEno4581 provided new perspectives on its potential roles in candidiasis (fungal-host interactions) caused by M. guilliermondii, especially M. guilliermondii strain SO on zebrafish embryos that mimic the immunocompromised individuals as previously evident.

In silico structural exploration of serine protease from a CTG-clade yeast Meyerozyma guilliermondii strain SO.

In eukaryotes, serine proteases are cellular localized hydrolases reported to regulate essential biological reactions. Improved industrial applications of proteins are aided by prediction and analysis of their 3-dimensional structures (3D). A serine protease was identified from CTG-clade yeast Meyerozyma guilliermondii strain SO and its 3D structure as well as its catalytic attributes have not been fully understood yet, thus we seek to report on the catalytic mechanism of M. guilliermondii strain SO MgPRB1 using substrate PMSF via in silico docking as well as its stability by way of disulfide bonds formation. Herein, bioinformatics tools and techniques were used to predict, validate and analyze the possible changes of CUG ambiguity (if any) in strain SO using template PDB ID: 3F7O. Structural assessments confirmed the classic catalytic triad Asp305, His337, and Ser499. Superimposition of MgPRB1 and template 3F7O structures revealed the unlinked cysteine residues between Cys341, Cys440, Cys471 and Cys506 of MgPRB1 compared to template 3F7O with two disulfide bonds formation, which confers structural stability. In conclusion, serine protease structure from strain SO was successfully predicted and studies towards understanding at the molecular level may be undertaken for its potential applications in the degradation of peptide bonds.

Whole genome sequencing and metabolomics analyses reveal the biosynthesis of nerol in a multi-stress-tolerant Meyerozyma guilliermondii GXDK6.

BACKGROUND: Nerol (C10H18O), an acyclic monoterpene, naturally presents in plant essential oils, and is used widely in food, cosmetics and pharmaceuticals as the valuable fragrance. Meanwhile, chemical synthesis is the only strategy for large-scale production of nerol, and the disadvantages of chemical synthesis greatly limit the production and its application. These defects drive the interests of researchers shift to the production of nerol by eco-friendly methods known as biosynthesis methods. However, the main technical bottleneck restricting the biosynthesis of nerol is the lacking of corresponding natural aroma-producing microorganisms. RESULTS: In this study, a novel multi-stress-tolerant probiotics Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified by whole genome sequencing and metabolomics technology. GXDK6 showed a broad pH tolerance in the range of 2.5-10.0. The species also showed salt tolerance with up to 12% NaCl and up to 18% of KCl or MgCl2. GXDK6 exhibited heavy-metal Mn2+ tolerance of up to 5494 ppm. GXDK6 could also ferment with a total of 21 kinds of single organic matter as the carbon source, and produce abundant aromatic metabolites. Results from the gas chromatography-mass spectrometry indicated the production of 8-14 types of aromatic metabolites (isopentanol, nerol, geraniol, phenylethanol, isobutanol, etc.) when GXDK6 was fermented up to 72 h with glucose, sucrose, fructose, or xylose as the single carbon source. Among them, nerol was found to be a novel aromatic metabolite from GXDK6 fermentation, and its biosynthesis mechanism had also been further revealed. CONCLUSION: A novel aroma-producing M. guilliermondii GXDK6 was identified successfully by whole genome sequencing and metabolomics technology. GXDK6 showed high multi-stress-tolerant properties with acid-base, salty, and heavy-metal environments. The aroma-producing mechanism of nerol in GXDK6 had also been revealed. These findings indicated the aroma-producing M. guilliermondii GXDK6 with multi-stress-tolerant properties has great potential value in the fermentation industry.

Biocontrol ability and action mechanisms of Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 against Penicillium digitatum DSM2750 and Penicillium expansum DSM62841 causing postharvest diseases.

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 106 and 108 cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 108 cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.

Clinical Characteristics and Outcomes of Candidemia Caused by Meyerozyma guilliermondii Complex in Cancer Patients Undergoing Surgery.

Although Meyerozyma guilliermondii complex is an uncommon cause of invasive candidiasis worldwide, reported cases, mainly regarding bloodstream infections, increased over years, and patients with cancer who have undergone recent surgery are most commonly affected. However, the clinical characteristics and outcomes of candidemia caused by M. guilliermondii complex remain poorly understood. A retrospective case-control study was conducted to evaluate the clinical characteristics and mortality of candidemia caused by M. guilliermondii complex in cancer patients undergoing surgery. Demographic and clinical data were collected from the hospital medical records system with a standardized data collection form and were analyzed with SPSS 20.0. Sixty-six cancer patients who have undergone recent surgery and were diagnosed with candidemia caused by M. guilliermondii complex were included in the study. Regarding the clinical manifestations, most patients' body temperatures ranged from 38 to 40 °C, with a median fever duration of 4 (IQR: 3-6) days. Multivariate analysis indicated that the presence of central venous catheter (OR: 6.68; 95% CI 2.80-15.94) and gastric tube (OR: 3.55; 95% CI 1.22-10.34) were independent risk factors for M. guilliermondii complex fungemia. The 30-day crude mortality of candidemia caused by M. guilliermondii complex was 12.1%, twice that of the control group. Moreover, increased WBC count, age ≥ 60 years, septic shock, and ICU admission were identified as predictors of mortality through univariate analysis. These findings will provide a foundation for the clinical management of candidemia caused by M. guilliermondii complex in post-surgical cancer patients.

[Toxic effects of AFB_1/T-2 toxin and intervention effects of Meyerozyma guilliermondii in dried Lutjanus erythopterus on mice].

OBJECTIVE: In order to investigate the effect of yeast on reducing mycotoxin damage in dried fish. METHODS: A strain of Meyerozyma guilliermondii MH 211588. 1(MG-81) was mixed and fermented 48 h with dried Lutjanus erythopterus which contaminated aflatoxin B_1(AFB_1) and T-2 toxin(T-2). The toxin concentration in fermentation at different time was detected by LC-MS/MS, and fermentation was fed with mice by intragastric administration(7 d). Blood routine and four liver function enzymes were measured by hematology analyzer and microplate spectrophotometer respectively. The elimination effect of MG-81 isolate on mycotoxin damage in dried fish was evaluated by the toxin concentration at different time and its toxic effect on mice. RESULTS: The removal rates of AFB_1 and T-2 in dried fish fermentation showed a parabolic linear growth trend with the prolongation of fermentation time. The removal rates of AFB_1 and T-2 in dried fish fermentation broth tended to be stable at 36 h(the removal rates of AFB_1 and T-2 were 83. 7%±1. 3% and 78. 5%±0. 8%). This indicated that 36 h was the optimal time for MG-81 to remove mycotoxins in dried fish. At the same time, it was found that there was no significant change in the indexes of MG-81 dried fish fermentation compared with the control group(P>0. 05), while the same dose of AFB_1 and T-2 dried fish fermentation(without MG-81), the leucocytes, lymphocytes, erythrocyte, hemoglobin, platelet and mean platelet volume of mice were significantly lower than those of control group(P>0. 05), showing obvious hemotoxicity and immunotoxicity. The activity of four liver enzymes was increased significantly(P<0. 05), showing obvious hepatotoxicity. CONCLUSION: The fermentation of MG-81 for 36 h can effectively remove AFB_1 and T-2 from dried fish and eliminate their hazards.

In vitro lytic activity and antifungal susceptibility of infrequently isolated yeasts.

Non-albicans Candida species have acquired relevance in the last decades as a cause of serious disease. The virulence factors and antifungal susceptibility of these rare pathogens remain largely unrecognized. We examined a total of 50 yeast isolates corresponding to 11 different infrequently isolated yeast species for their in vitro enzymatic profile and susceptibility pattern as first-line antifungals. We found aspartyl protease activity for 100% of the isolates tested as well as variable DNAse, hemolysin, phospholipase and esterase activities. All strains had low MICs for amphotericin B and showed a variable response to fluconazole (0.125-32 µg/mL) and the echinocandins tested (0.25-> 8 µg/mL).

Process parameters for biosurfactant production using yeast Meyerozyma guilliermondii YK32.

Microbially produced biosurfactants are fast catching up due to their environment-friendly approach over chemical surfactants. But their commercial production is restricted due to poor economy of the production process which could be improved by using high yielding microbial strains and optimizing the process parameters. The present research was directed to optimize the biosurfactant production monitored in terms of oil displacement and emulsification (E24) index, using a promising yeast Meyerozyma guilliermondii YK32. Maximum oil displacement equaling 7.5 cm was obtained with olive oil at 8% (v/v) concentration as carbon source under shaking conditions (150 rpm). Diesel being a complex hydrocarbon was not utilized easily by yeast and showed poor biosurfactant production. Yeast extract at 1.5% (w/v) concentration yielded maximum biosurfactant as evident from maximum oil displacement and E24 index equal to 8.1 cm and 52.6%, respectively. Sodium chloride at the rate of 3% (w/v) supported maximum oil displacement (8.8 cm) using the production broth containing optimized carbon and nitrogen sources. Any increase beyond this level negatively influenced the biosurfactant production. The yield was at its maximum at 30 °C as a shift in temperature either to 35 °C or 25 °C decreased the oil displacement from 8.8 to 5.2 or 7.6 cm, respectively. At 40 °C, oil displacement was decreased to 2.5 cm. Biosurfactant production appeared to be sensitive to varying pH as evident from the E24 index as high as 67.3% at pH 6.0 as compared with 60.2%, 60.1%, and 52.4% at pH 5.0, 5.5, and 7.0, respectively. Yeast biomass yield equivalent to 10.3 g/L and 8.3 g/L was recorded at pH 6 and 7, respectively, during the production process. Elimination of shaking reduced the E24 index from 67.3 to 34.8% under optimized conditions.

LSP1, a responsive protein from Meyerozyma guilliermondii, elicits defence response and improves glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis Fisch adventitious roots.

This research explored the effects of protein and polysaccharide in Meyerozyma guilliermondii on active compounds in Glycyrrhiza uralensis Fisch adventitious roots. In this study, a responsive protein LSP1 was purified from the Meyerozyma guilliermondii since the excellent induction. The contents of total flavonoids (3.46 mg · g-1 ), glycyrrhizic acid (0.41 mg · g-1 ), glycyrrhetinic acid (0.41 mg · g-1 ), and polysaccharide (94.49 mg · g-1 ) in adventitious root peaked at LSP1 group, which were 1.6, 3.4, 2.4, 2.0-fold that of control, respectively. Besides, the responsive protein LSP1 significantly activated the defense signaling, mitogen-activated protein kinases and extremely up-regulated the expression of defense-related genes and functional genes involved in glycyrrhizic acid biosynthesis.

Gene expression of glycyrrhizin acid and accumulation of endogenous signaling molecule in Glycyrrhiza uralensis Fisch adventitious roots after Saccharomyces cerevisiae and Meyerozyma guilliermondii applications.

This study reports the best culture conditions for roots growth and accumulation of active components by optimizing the parameters. Glycyrrhiza uralensis adventitious roots metabolites were significantly increased after adding Saccharomyces cerevisiae and Meyerozyma guilliermondii. The highest contents of polysaccharide, glycyrrhizic acid, glycyrrhetinic acid, and total flavonoids were obtained in M. guilliermondii group; the content of glycyrrhizic acid was 5.3-fold higher than the control. In control and treatment groups, 12 compounds were identified by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), among which some new compounds have been detected in elicitor groups including 5,7-dihydroxyflavanone, glycyrrhisoflavanone, licorice saponin J2, uralsaponin B, (3R)-vestitol, and uralenol. Meyerozyma guilliermondii significantly upregulated the expression of the genes such as 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, geranyl diphosphate synthase, squalene synthase, squalene epoxidase, ß-amyrin synthase, and CYP88D6 and CYP72A154. Meanwhile, it increased the biosynthesis of signaling molecules (nitric oxide, salicylic acid, and jasmonic acid) in defense mechanism.

Nitrile-hydrolyzing enzyme from Meyerozyma guilliermondii and its potential in biosynthesis of 3-hydroxypropionic acid.

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag+, Pb2+ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

Phenotypic and molecular diversity of Meyerozyma guilliermondii strains isolated from food and other environmental niches, hints for an incipient speciation.

Meyerozyma guilliermondii is a yeast species widely isolated from several natural environments and from fruit; in medical microbiology it is known as the teleomorph of the opportunistic pathogen Candida guilliermondii, which causes about 2% of the human blood infections. This yeast is also promising in a variety of biotechnological applications as vitamins production and post-harvest control. The question if isolates from different sources are physiologically and genetically similar, or if the various environments induced significant differences, is crucial for the understanding of this species structure and to select strains appropriate for each application. This question was addressed using LSU and ITS sequencing for taxonomic assignment, i-SSR (GACA4) for the molecular characterization and FTIR for the metabolomic fingerprint. All data showed that fruit and environmental isolates cluster separately with a general good agreement between metabolomics and molecular analysis. An additional RAPD analysis was able to discriminate strains according to the isolation position within the pineapple fruit. Although all strains are members of the M. guilliermondii species according to the current standards, the distribution of large variability detected suggests that some specialization occurred in the niches inhabited by this yeast and that food related strains can be differentiated from the medical isolates.

New depsidones and isoindolinones from the mangrove endophytic fungus Meyerozyma guilliermondii (HZ-Y2) isolated from the South China Sea.

Three new depsidones, botryorhodines E-G (1-3), and two new isoindolinones, meyeroguillines A and B (7 and 9), along with five known compounds were isolated from an endophytic fungus Meyerozyma guilliermondii, derived from the mangrove plant Kandelia obovata. Their structures were elucidated by 1D and 2D NMR spectroscopy and high resolution mass spectrometry (HREIMS). Compounds 1-6 exhibited strong α-glucosidase inhibitory activity with IC50 values ranging from 2.1 to 13.3 µM. Moreover, kinetic studies of compounds 2 and 6 showed that both of them were noncompetitive inhibitors of α-glucosidase.

Regulatory mechanisms of dopamine metabolism in a marine Meyerozyma guilliermondii GXDK6 under NaCl stress as revealed by integrative multi-omics analysis.

Dopamine can be used to treat depression, myocardial infarction, and other diseases. However, few reports are available on the de novo microbial synthesis of dopamine from low-cost substrate. In this study, integrated omics technology was used to explore the dopamine metabolism of a novel marine multi-stress-tolerant aromatic yeast Meyerozyma guilliermondii GXDK6. GXDK6 was found to have the ability to biosynthesize dopamine when using glucose as the substrate. 14 key genes for the biosynthesis of dopamine were identified by whole genome-wide analysis. Transcriptomic and proteomic data showed that the expression levels of gene AAT2 encoding aspartate aminotransferase (regulating dopamine anabolism) were upregulated, while gene AO-I encoding copper amine oxidase (involved in dopamine catabolism) were downregulated under 10 % NaCl stress compared with non-NaCl stress, thereby contributing to biosynthesis of dopamine. Further, the amount of dopamine under 10 % NaCl stress was 2.51-fold higher than that of zero NaCl, which was consistent with the multi-omics results. Real-time fluorescence quantitative PCR (RT-qPCR) and high-performance liquid chromatography (HPLC) results confirmed the metabolic model of dopamine. Furthermore, by overexpressing AAT2, AST enzyme activity was increased by 24.89 %, the expression of genes related to dopamine metabolism was enhanced, and dopamine production was increased by 56.36 % in recombinant GXDK6AAT2. In conclusion, Meyerozyma guilliermondii GXDK6 could utilize low-cost carbon source to synthesize dopamine, and NaCl stress promoted the biosynthesis of dopamine.

Features of the rare pathogen Meyerozyma guilliermondii strain SO and comprehensive in silico analyses of its adherence-contributing virulence factor agglutinin-like sequences.

Meyerozyma guilliermondii is a rare yeast pathogen contributing to the deadly invasive candidiasis. M. guilliermondii strain SO, as a promising protein expression host, showed 99% proteome similarity with the clinically isolated ATCC 6260 (type strain) in a recent comparative genomic analysis. However, their in vitro virulence features and in vivo pathogenicity were uncharacterized. This study aimed to characterize the in vitro and in vivo pathogenicity of M. guilliermondii strain SO and analyze its Als proteins (MgAls) via comprehensive bioinformatics approaches. M. guilliermondii strain SO showed lower and higher sensitivity towards ß-mercaptoethanol and lithium, respectively than the avirulent S. cerevisiae but exhibited the same tolerance towards cell wall-perturbing Congo Red with C. albicans. With 7.5× higher biofilm mass, M. guilliermondii strain SO also demonstrated 75% higher mortality rate in the zebrafish embryos with a thicker biofilm layer on the chorion compared to the avirulent S. cerevisiae. Being one of the most important Candida adhesins, sequence and structural analyses of four statistically identified MgAls showed that MgAls1056 was predicted to exhibit the most conserved amyloid-forming regions, tandem repeat domain and peptide binding cavity (PBC) compared to C. albicans Als3. Favoured from the predicted largest ligand binding site and druggable pockets, it showed the highest affinity towards hepta-threonine. Non-PBC druggable pockets in the most potent virulence contributing MgAls1056 provide new insights into developing antifungal drugs targeting non-albicans Candida spp. Virtual screening of available synthetic or natural bioactive compounds and MgAls1056 deletion from the fungal genome should be further performed and validated experimentally.Communicated by Ramaswamy H. Sarma.

Yeast Diversity in Honey and Pollen Samples from Stingless Bees in the State of Bahia, Brazil: Use of the MALDI-TOF MS/Genbank Proteomic Technique.

(1) Background: The identification of microorganisms includes traditional biochemical methods, molecular biology methods evaluating the conserved regions of rRNA, and the molecular biology of proteins (proteomics), such as MALDI-TOF MS mass spectrometry. This work aimed to identify the biodiversity of yeasts associated with stingless bee species' honey and pollen, Melipona scutellaris, Nannotrigona testaceicornes, and Tetragonisca angustula, from the region of São Gonçalo dos Campos-Bahia (BA) state, Brazil. (2) Methods: Cellular proteins were extracted from 2837 microbial isolates (pollen and honey) and identified via MALDI-TOF MS. The identified yeast species were also compared to the mass spectra of taxonomically well-characterized reference strains, available from the National Center of Biotechnology Information (NCBI) database. (3) Results: Nine yeast species were identified: Candida maltosa, Candida norvegica, Kazachstania telluris, Schizosaccharomyces pombe, Scheffersomyces insectosus, Meyerozyma guilliermondii, Brettanomyces bruxellensis, Kazachstania exigua, and Starmerella lactis-condensi. Nannotrigona testaceicornes pollen had the highest number of yeast colonies. The yeasts Brettanomyces bruxellensis and Kazachstania telluris showed high populations in the samples of Nannotrigona testaceicornes and Melipona scutellaris, respectively. This work shows that there is some sharing of the same species of yeast between honey and pollen from the same beehive. (4) Conclusions: A total of 71.84% of the identified species present a high level of confidence at the species level. Eight yeast species (Candida maltosa, Candida norvegica, Kazachstania telluris, Schizosaccharomyces pombe, Scheffersomyces insectosus, Meyerozyma guilliermondii, Kazachstania exigua, and Starmerella lactis-condensi) were found for the first time in the samples that the authors inspected. This contributes to the construction of new knowledge about the diversity of yeasts associated with stingless bee products, as well as to the possibility of the biotechnological application of some yeast species.