Genomic and functional analyses unveil the response to hyphal wall stress in Candida albicans cells lacking ß(1,3)-glucan remodeling.

BACKGROUND: The cell wall is essential for the yeast to hypha (Y-H) transition that enables Candida albicans to invade human tissues and evade the immune system. The main constituent, ß(1,3)-glucan, is remodeled by glucanosyltransferases of the GH72 family. Phr1p is responsible of glucan remodeling at neutral-alkaline pH and is essential for morphogenesis and virulence. Due to the pH-regulated expression of PHR1, the phr1Δ phenotype is manifested at pH > 6 and its severity increases with the rise in pH. We exploited the pH-conditional nature of a PHR1 null mutant to analyze the impact of glucan remodeling on the hyphal transcriptional program and the role of chitin synthases in the hyphal wall stress (HWS) response. RESULTS: In hyphal growth inducing conditions, phr1Δ germ tubes are defective in elongation, accumulate chitin, and constitutively activate the signaling pathways mediated by the MAP kinases Mkc1p, Cek1p and Hog1p. The transcriptional profiles revealed an increase of transcript levels for genes involved in cell wall formation (CHS2 and CHS8, CRH11, PGA23, orf19.750, RBR1, RBT4, ECM331, PGA6, PGA13), protein N-glycosylation and sorting in the ER (CWH8 and CHS7), signaling (CPP1, SSK2), ion transport (FLC2, YVC1), stress response and metabolism and a reduced expression of adhesins. A transient up-regulation of DNA replication genes associated with entry into S-phase occurred whereas cell-cycle regulating genes (PCL1, PCL2, CCN1, GIN4, DUN1, CDC28) were persistently up-regulated. To test the physiological relevance of altered CHS gene expression, phr1Δ chsxΔ (x = 2,3,8) mutant phenotypes were analyzed during the Y-H transition. PHR1 deletion was synthetic lethal with CHS3 loss on solid M199 medium-pH 7.5 and with CHS8 deletion on solid M199-pH 8. On Spider medium, PHR1 was synthetic lethal with CHS3 or CHS8 at pH 8. CONCLUSIONS: The absence of Phr1p triggers an adaptive response aimed to reinforce the hyphal cell wall and restore homeostasis. Chs3p is essential in preserving phr1Δ cell integrity during the Y-H transition. Our findings also unveiled an unanticipated essential role of Chs8p during filamentation on solid media. These results highlight the flexibility of fungal cells in maintaining cell wall integrity and contribute to assessments of glucan remodeling as a target for therapy.

Acetaldehyde as an Intermediate in the Electroreduction of Carbon Monoxide to Ethanol on Oxide-Derived Copper.

Oxide-derived copper (OD-Cu) electrodes exhibit unprecedented CO reduction performance towards liquid fuels, producing ethanol and acetate with >50% Faradaic efficiency at -0.3 V (vs. RHE). By using static headspace-gas chromatography for liquid phase analysis, we identify acetaldehyde as a minor product and key intermediate in the electroreduction of CO to ethanol on OD-Cu electrodes. Acetaldehyde is produced with a Faradaic efficiency of ≈5% at -0.33 V (vs. RHE). We show that acetaldehyde forms at low steady-state concentrations, and that free acetaldehyde is difficult to detect in alkaline solutions using NMR spectroscopy, requiring alternative methods for detection and quantification. Our results represent an important step towards understanding the CO reduction mechanism on OD-Cu electrodes.

Major contribution of the Ehrlich pathway for 2-phenylethanol/rose flavor production in Ashbya gossypii.

Aroma alcohols of fermented food and beverages are derived from fungal amino acids catabolism via the Ehrlich pathway. This linear pathway consists of three enzymatic reactions to form fusel alcohols. Regulation of some of the enzymes occurs on the transcriptional level via Aro80. The riboflavin overproducer Ashbya gossypii produces strong fruity flavours in contrast to its much less aromatic relative Eremothecium cymbalariae. Genome comparisons indicated that A. gossypii harbors genes for aromatic amino acid catabolism (ARO8a, ARO8b, ARO10, and ARO80) while E. cymbalariae only encodes ARO8a and thus lacks major components of aromatic amino acid catabolism. Volatile compound (VOC) analysis showed that both Eremothecium species produce large amounts of isoamyl alcohol while A. gossypii also produces high levels of 2-phenylethanol. Deletion of the A. gossypii ARO-genes did not confer any growth deficiencies. However, A. gossypii ARO-mutants (except Agaro8a) were strongly impaired in aroma production, particularly in the production of the rose flavour 2-phenylethanol. Conversely, overexpression of ARO80 via the AgTEF1 promoter resulted in 50% increase in VOC production. Together these data indicate that A. gossypii is a very potent flavour producer and that amongst the non-Saccharomyces biodiversity strains can be identified that could provide positive sensory properties to fermented beverages.

Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae.

Production of plant metabolites in microbial hosts represents a promising alternative to traditional chemical-based methods. Diterpenoids are compounds with interesting applications as pharmaceuticals, fragrances and biomaterials. Casbene, in particular, serves as a precursor to many complex diterpenoids found in plants from the Euphorbiaceae family that have shown potential therapeutic effects. Here, we engineered the budding yeast Saccharomyces cerevisiae for improved biosynthesis of the diterpene casbene. We first expressed, in yeast, a geranylgeranyl diphosphate synthase from Phomopsys amygdali in order to boost the geranylgeranyl diphosphate pool inside the cells. The enzyme uses isopentenyl diphosphate and dimethylallyl diphosphate to directly generate geranylgeranyl diphosphate. When co-expressing a casbene synthase from Ricinus communis the yeast was able to produce casbene in the order of 30 mg/L. Redirecting the flux from FPP and sterols, by means of the ergosterol sensitive promoter of ERG1, allowed for plasmid-based casbene production of 81.4 mg/L. Integration of the target genes into the yeast genome, together with the replacement of the promoter regions of ERG20 and ERG9 with combinations of ergosterol- and glucose-sensitive promoters, generated a titer of 108.5 mg/L of casbene. We here succeeded to engineer an improved route for geranylgeranyl diphosphate synthesis in yeast. Furthermore, we showed that the concurrent dynamic control of ERG20 and ERG9 expression, using ergosterol and carbon source regulation mechanisms, could substantially improve diterpene titer. Our approach will pave the way for a more sustainable production of GGPP- and casbene-derived products.

Development of brewing science in (and since) the late 19th century: molecular profiles of 110-130year old beers.

The 19th century witnessed many advances in scientific enzymology and microbiology that laid the foundations for modern biotechnological industries. In the current study, we analyze the content of original lager beer samples from the 1880s, 1890s and 1900s with emphasis on the carbohydrate content and composition. The historic samples include the oldest samples brewed with pure Saccharomyces carlsbergensis yeast strains. While no detailed record of beer pasteurization at the time is available, historic samples indicate a gradual improvement of bottled beer handling from the 1880s to the 1900s, with decreasing contamination by enzymatic and microbial activities over this time span. Samples are sufficiently well preserved to allow comparisons to present-day references, thus yielding molecular signatures of the effects of 20th century science on beer production. Opposite to rather stable carbohydrate profiles, some aldehydes reach up to 40-fold higher levels in the historic samples as compared to present-day references.

An indirect assay for volatile compound production in yeast strains.

Traditional flavor analysis relies on gas chromatography coupled to mass spectrometry (GC-MS) methods. Here we describe an indirect method coupling volatile compound formation to an ARO9-promoter-LacZ reporter gene. The resulting ß-galactosidase activity correlated well with headspace solid phase micro extraction (HS/SPME) GC-MS data, particularly with respect to the formation of rose flavor. This tool enables large-scale screening of yeast strains and their progeny to identify the most flavor active strains.