Metabolomics-Guided Analysis of the Biocatalytic Conversion of Sclareol to Ambradiol by Hyphozyma roseoniger

The biocatalytic conversion of sclareol to ambradiol, a valuable component in the fragrance industry, using whole-cell biotransformation by the dimorphic yeast Hyphozyma roseoniger, was investigated using metabolomics tools. An integrated approach was used to identify and quantify the participating intermediates in this bioconversion using both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to mass spectrometry (LC–MS). This study entailed growth stage-dependent analysis of H. roseoniger suspensions grown in batch culture over a 14-day period, beginning with a three-day induction period using 20 mg/200 mL sclareol, followed by a further 1 g/200 mL sclareol dose to enable ambradiol production. The progress of the bioconversion and the resulting dynamic changes to the metabolome were monitored using NMR analysis and semi-targeted LC–MS metabolomics. This outlined the molecular conversions occurring within the matrix and no novel intermediates participating in the sclareol to ambradiol conversion could be identified. This study presents new findings about the transformative capabilities of H. roseoniger as a whole cell biocatalyst, highlighting its potential utility in similar applications.

CO2 accelerates Candida albicans biofilm formation and reveals novel approaches to their inhibition on airway management devices

C. albicans is the predominant human fungal pathogen worldwide and frequently colonises medical devices, such as voice prosthesis, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance the effects of high CO2 levels on C. albicans biofilm growth has not been investigated to date. Here, we show that 5% CO2 significantly enhances each stage of the C. albicans biofilm forming process; from attachment through maturation to dispersion, via stimulation of the Ras/cAMP/PKA signalling pathway. Transcriptome analysis of biofilm formation under elevated CO2 conditions revealed the activation of key biofilm formation pathways governed by the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown in under elevated CO2 conditions also exhibit increases in azole resistance, tolerance to nutritional immunity and enhanced glucose uptake capabilities. We thus characterise the mechanisms by which elevated CO2 promote C. albicans biofilm formation. We also investigate the possibility of re-purposing drugs that can target the CO2 activated metabolic enhancements observed in C. albicans biofilms. Using this approach we can significantly reduce multi-species biofilm formation in a high CO2 environment and demonstrate a significant extension of the lifespan of voice prostheses in a patient trial. Our research demonstrates a bench to bedside approach to tackle Candida albicans biofilm formation.

A Taphrina strain infecting the model plant Arabidopsis thaliana.

Yeasts are important plant-associated organisms that can modulate host immunity to either promote or prevent disease. Mechanisms of plant-yeast interactions, specifically of yeast perception by the plant innate immune system, remain unknown. Progress has been hindered by the scarcity of yeast species associated with the model plant Arabidopsis thaliana (Arabidopsis). We have previously isolated Taphrina strain M11 from wild Arabidopsis in the field. Taphrina are poorly studied dimorphic yeast-like fungi that are plant pathogens, often producing plant hormones and causing tumour-like leaf deformation symptoms on their hosts. Here we characterize the interaction of M11 with Arabidopsis. Infection of Arabidopsis with the birch pathogen T. betulina, used as a non-host control, shows early HR, enhanced ROS accumulation, and limitation of growth, demonstrating that Arabidopsis has immunity against non-adapted yeasts. M11 triggered limited cell death, an attenuated ROS response, and grew in planta, as well as subtle but clear leaf deformation symptoms, demonstrating it is pathogenic. Hormone responsive promoter-reporter analysis demonstrated activation of cytokinin signalling during infection. Mutant infection assays indicate jasmonate and ethylene were required for immunity against M11. Analysis of the Taphrina M11 genome was used to mine evidence for yeast specific PAMPs which may underlie host immune responses against yeast-like fungi.

CO2 enhances the formation, nutrient scavenging and drug resistance properties of C. albicans biofilms

C. albicans is the predominant human fungal pathogen and frequently colonises medical devices, such as voice prostheses, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is the elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance, the effect of CO2 on C. albicans biofilm growth has not been investigated to date. Here we show that physiologically relevant CO2 elevation enhances each stage of the C. albicans biofilm-forming process: from attachment through maturation to dispersion. The effects of CO2 are mediated via the Ras/cAMP/PKA signalling pathway and the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown under elevated CO2 conditions also exhibit increased azole resistance, increased Sef1-dependent iron scavenging and enhanced glucose uptake to support their rapid growth. These findings suggest that C. albicans has evolved to utilise the CO2 signal to promote biofilm formation within the host. We investigate the possibility of targeting CO2-activated processes and propose 2-deoxyglucose as a drug that may be repurposed to prevent C. albicans biofilm formation on medical airway management implants. We thus characterise the mechanisms by which CO2 promotes C. albicans biofilm formation and suggest new approaches for future preventative strategies.

The pH-Responsive Transcription Factors YlRim101 and Mhy1 Regulate Alkaline pH-Induced Filamentation in the Dimorphic Yeast Yarrowia lipolytica

ABSTRACT Environmental pH influences cell growth and differentiation. In the dimorphic yeast Yarrowia lipolytica, neutral-alkaline pH strongly induces the yeast-to-filament transition. However, the regulatory mechanism that governs alkaline pH-induced filamentation has been unclear. Here, we show that the pH-responsive transcription factor Y. lipolytica Rim101 (YlRim101) is a major regulator of alkaline-induced filamentation, since the deletion of YlRIM101 severely impaired filamentation at alkaline pH, whereas the constitutively active YlRIM1011-330 mutant mildly induced filamentation at acidic pH. YlRim101 controls the expression of the majority of alkaline-regulated cell wall protein genes. One of these, the cell surface glycosidase gene YlPHR1, plays a critical role in growth, cell wall function, and filamentation at alkaline pH. This finding suggests that YlRim101 promotes filamentation at alkaline pH via controlling the expression of these genes. We also show that, in addition to YlRim101, the Msn2/Msn4-like transcription factor Mhy1 is highly upregulated at alkaline pH and is essential for filamentation. However, unlike YlRim101, which specifically regulates alkaline-induced filamentation, Mhy1 regulates both alkaline- and glucose-induced filamentation, since the deletion of MHY1 abolished them both, whereas the overexpression of MHY1 induced strong filamentation irrespective of the pH or the presence of glucose. Finally, we show that YlRim101 and Mhy1 positively coregulate seven cell wall protein genes at alkaline pH, including YlPHR1 and five cell surface adhesin-like genes, three of which appear to promote filamentation. Together, these results reveal a conserved role of YlRim101 and a novel role of Mhy1 in the regulation of alkaline-induced filamentation in Y. lipolytica. IMPORTANCE The regulatory mechanism that governs pH-regulated filamentation is not clear in dimorphic fungi except in Candida albicans. Here, we investigated the regulation of alkaline pH-induced filamentation in Yarrowia lipolytica, a dimorphic yeast distantly related to C. albicans. Our results show that the transcription factor YlRim101 and the Msn2/Msn4-like transcription factor Mhy1 are the major regulators that promote filamentation at alkaline pH. They control the expression of a number of cell wall protein genes important for cell wall organization and filamentation. Our results suggest that the Rim101/PacC homologs play a conserved role in pH-regulated filamentation in dimorphic fungi.

CO2 enhances the ability of Candida albicans to form biofilms, overcome nutritional immunity and resist antifungal treatment

C. albicans is the predominant fungal pathogen of humans and frequently colonises medical devices, such as voice prosthesis, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is the elevation of CO2 levels, as observed in exhaled breath.. However, despite the clear medical relevance, the effects of CO2 on C. albicans biofilm growth has not been investigated to date. Here, we show that physiologically relevant CO2 elevation enhances each stage of the C. albicans biofilm forming process;from attachment through to maturation and dispersion.. The effects of CO2 are mediated via the Ras/cAMP/PKA signalling pathway and the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown under elevated CO2 conditions also exhibit increased azole resistance, tolerance to nutritional immunity and enhanced glucose uptake to support their rapid growth. These findings suggest that C. albicans has evolved to utilise the CO2 signal to promote biofilm formation within the host. We investigate the possibility of targeting CO2 activated processes and propose 2-Deoxyglucose as a drug that may be repurposed to prevent C. albicans biofilm formation on medical airway management implants. We thus characterise the mechanisms by which CO2 promotes C. albicans biofilm formation and suggest new approaches for future preventative strategies.

Molecular studies of NAD- and NADP- glutamate dehydrogenases decipher the conundrum of yeast-hypha dimorphism in zygomycete Benjaminiella poitrasii

Benjaminiella poitrasii, a zygomycete shows glucose and temperature dependent yeast (Y)-hypha (H) dimorphic transition. Earlier we reported the biochemical correlation of relative proportion of NAD- and NADP- glutamate dehydrogenases (GDH) with Y-H transition. Further, we observed the presence of one NAD-GDH and two form–specific NADP-GDH isoenzymes in B. poitrasii. However, molecular studies are necessary to elucidate the explicit role of GDHs in regulating Y-H reversible transition. Here, we report the isolation and characterization of one NAD- (BpNADGDH, 2.643 kb) and two separate genes, BpNADPGDH I (Y form specific, 1.365 kb) and BpNADPGDH II (H form specific, 1.368 kb) coding for NADP-GDH isoenzymes in B. poitrasii. The transcriptional profiling during Y-H transition showed higher BpNADPGDH I expression in Y cells while expression of BpNADPGDH II was higher in H cells. Moreover, the yeast form monomorphic mutant (Y-5) did not show BpNADPGDH II expression under normal dimorphism triggering conditions. Transformation with H form specific BpNADPGDH II induced the germ tube formation in Y-5, which confirmed the cause-effect relationship between BpNADPGDH genes and morphological outcome in B. poitrasii. Interestingly, expression of H-form specific BpNADPGDH II also induced germ tube formation in human pathogenic, non-dimorphic yeast Candida glabrata, which further corroborated our findings.

Phytochemical Investigations of Laurel Fruits (Laurus nobilis)

Bay laurel ( Laurus nobilis L.) is an evergreen tree. The objective of this study was to determine the chemical composition (polyphenols, essential oil [EO], lipid fraction, cellulose, and protein content) of laurel fruits collected from Greece (Mount Athos) and Georgia (the village of Meria), and to evaluate the antimicrobial activity of laurel fruit EOs. The major phenolic acids in the fruits from Greece were p-coumaric acid (free 261.6 µg/g) and vanillic acid (free 253.1 µg/g and conjugated 925.8 µg/g). The major phenolic acids in fruits from Georgia were vanillic acid (free 105.6 µg/g and caffeic acid [conjugated 439.2 µg/g], and syringic acid [conjugated 390.7 µg/g]). The laurel fruit EOs from Greece (1.4% content) and Georgia (1.6%) had distinct composition. Monoterpene hydrocarbons were the dominant group of compounds in the EOs, with 49.7% in the EO from Greece and 68.7% in the EO from Georgia. The major constituents of the fruit EO from Greece were 1,8-cineole (18.2%), α-phellandrene (15.0 %), β-pinene (9.4%), and α -pinene (9.1%), whereas the ones from Georgia were trans-β-ocimene (59.4%) and 1,8-cineole (7.6%). Laurel fruit EO from Greece and Georgia demonstrated low to moderate antimicrobial activity against pathogenic and spoilage microorganisms and the dimorphic yeast Candida albicans. The main fatty acids (FAs) in the lipid fractions were oleic, palmitic, and linoleic; there were differences in FA composition between the shells and the seeds of the fruits from the two countries. γ-Тocopherol predominated in the tocopherol fraction of the lipids from fruits shells and seeds from Greece (65.3% and 54.4%, respectively), whereas β-tocopherol predominated in fruits shells and seeds from Georgia (93.7% and 45.6%, respectively). Currently underutilized, the laurel fruits from both Greece and Georgia contain various valuable compounds that may potentially be used for perfumery, cosmetic, and pharmaceutical applications.