Glycerol Biosynthesis Research Articles

The Genome-Wide Mutation Shows the Importance of Cell Wall Integrity in Growth of the Psychrophilic Yeast Metschnikowia australis W7-5 at Different Temperatures.

In this study, it was found that a Cre/loxP system could be successfully used as a tool for editing the genome of the psychrophilic yeast Metschnikowia australis W7-5 isolated from Antarctica. The deletion and over-expression of the TPS1 gene for trehalose biosynthesis, the GSY gene for glycogen biosynthesis, and the GPD1 and GPP genes for glycerol biosynthesis had no influence on cell growth of the mutants and transformants compared to cell growth of their wild-type strain M. australis W7-5, indicating that trehalose, glycogen, and glycerol had no function in growth of the psychrophilic yeast at different temperatures. However, removal of the SLT2 gene encoding the mitogen-activated protein kinase in the cell wall integrity (CWI) signaling pathway and the SWI4 and SWI6 genes encoding the transcriptional activators Swi4/6 had the crucial influence on cell growth of the psychrophilic yeast at the low temperature, especially at 25 °C and expression of the genes related to cell wall and lipid biosynthesis. Therefore, the cell wall could play an important role in growth of the psychrophilic yeast at different temperatures and biosynthesis of cell wall was actively regulated by the CWI signaling pathway. This was the first time to show that the genome of the psychrophilic yeast was successfully edited and the molecular evidences were obtained to elucidate mechanisms of low temperature growth of the psychrophilic yeast from Antarctica.

C. elegans possess a general program to enter cryptobiosis that allows dauer larvae to survive different kinds of abiotic stress

All organisms encounter abiotic stress but only certain organisms are able to cope with extreme conditions and enter into cryptobiosis (hidden life). Previously, we have shown that C. elegans dauer larvae can survive severe desiccation (anhydrobiosis), a specific form of cryptobiosis. Entry into anhydrobiosis is preceded by activation of a set of biochemical pathways by exposure to mild desiccation. This process called preconditioning induces elevation of trehalose, intrinsically disordered proteins, polyamines and some other pathways that allow the preservation of cellular functionality in the absence of water. Here, we demonstrate that another stress factor, high osmolarity, activates similar biochemical pathways. The larvae that acquired resistance to high osmotic pressure can also withstand desiccation. In addition, high osmolarity significantly increases the biosynthesis of glycerol making larva tolerant to freezing. Thus, to survive abiotic stress, C. elegans activates a combination of genetic and biochemical pathways that serve as a general survival program.

Cellulase-added cassava ethanol process boosts ethanol titer and reduces glycerol production

Cassava is a widely used raw material for bioethanol production. In addition to starch, there is about 4–5 % (w/w) cellulose in cassava tuber, which is not utilized in current cassava ethanol process. Here, a bolt-on cellulase-added ethanol process was developed to realize the in situ conversion of cellulose in cassava tuber. As a result, the final ethanol titer increased from 127.94 g/L to 131.84 g/L, with 57.17 % cellulose and some recalcitrant starch additionally hydrolyzed. Moreover, the titer of glycerol, a main by-product of ethanol fermentation, decreased from 10.78 g/L to 9.95 g/L and the ratio of ethanol/glycerol increased from 11.87 to 13.25. To investigate the reason for the decreased glycerol production in this cellulase-added cassava ethanol process, the effect of medium viscosity on glycerol production was extensively investigated, which indicated, for the first time, that the reduction of glycerol biosynthesis was due to decreased viscosity aroused by the cellulase addition. Overall, this cellulase-added cassava ethanol process not only realized the conversion of cassava cellulose and higher ethanol titer, but also reduced the production of glycerol.

Hepatic transcriptome of the freeze-tolerant Cope\u2019s gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation and freezing

BackgroundCope’s gray treefrog, Dryophytes chrysoscelis, withstands the physiological challenges of corporeal freezing, partly by accumulating cryoprotective compounds of hepatic origin, including glycerol, urea, and glucose. We hypothesized that expression of genes related to cryoprotectant mobilization and stress tolerance would be differentially regulated in response to cold. Using high-throughput RNA sequencing (RNA-Seq), a hepatic transcriptome was generated for D. chrysoscelis, and gene expression was compared among frogs that were warm-acclimated, cold-acclimated, and frozen.ResultsA total of 159,556 transcripts were generated; 39% showed homology with known transcripts, and 34% of all transcripts were annotated. Gene-level analyses identified 34,936 genes, 85% of which were annotated. Cold acclimation induced differential expression both of genes and non-coding transcripts; freezing induced few additional changes. Transcript-level analysis followed by gene-level aggregation revealed 3582 differentially expressed genes, whereas analysis at the gene level revealed 1324 differentially regulated genes. Approximately 3.6% of differentially expressed sequences were non-coding and of no identifiable homology. Expression of several genes associated with cryoprotectant accumulation was altered during cold acclimation. Of note, glycerol kinase expression decreased with cold exposure, possibly promoting accumulation of glycerol, whereas glucose export was transcriptionally promoted by upregulation of glucose-6-phosphatase and downregulation of genes of various glycolytic enzymes. Several genes related to heat shock protein response, DNA repair, and the ubiquitin proteasome pathway were upregulated in cold and frozen frogs, whereas genes involved in responses to oxidative stress and anoxia, both potential sources of cellular damage during freezing, were downregulated or unchanged.ConclusionOur study is the first to report transcriptomic responses to low temperature exposure in a freeze-tolerant vertebrate. The hepatic transcriptome of Dryophytes chrysoscelis is responsive to cold and freezing. Transcriptomic regulation of genes related to particular pathways, such as glycerol biosynthesis, were not all regulated in parallel. The physiological demands associated with cold and freezing, as well as the transcriptomic responses observed in this study, are shared with several organisms that face similar ecophysiological challenges, suggesting common regulatory mechanisms. The role of transcriptional regulation relative to other cellular processes, and of non-coding transcripts as elements of those responses, deserve further study.

Биологическая роль глицерина в клетках дрожжей. Дрожжи как продуценты глицерина

This review contains information about the physiological role of glycerol as an osmoprotective and cryoprotective factor in the vital activity of yeast cells. The significance of the glycerol biosynthesis in yeast when cultured under anaerobic and microaerophilic conditions is shown. The dependence of the glycerol production by yeast on the cultivation conditions and the composition of nutrient media is discussed. The publications on glycerol production by various taxonomic yeast groups have been analyzed. Based on the studied literature material, prospects of using yeast organisms as producers of glycerol by fermentation are predicted. glycerol, yeast, hyperosmotic stress, fermentation

The unusual di-domain structure of Dunaliella salina glycerol-3-phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

Effect of Myclobutanil Pesticide on the Physiological Behavior of Two Newly Isolated Saccharomyces cerevisiae Strains during Very-High-Gravity Alcoholic Fermentation.

Yeasts are able to act as biosorbents, as their cell wall includes several components capable of binding organic xenobiotic compounds that can potentially be removed during various fermentation processes. In the present investigation, two novel Saccharomyces cerevisiae strains (LMBF-Y 16 and LMBF-Y-18), previously isolated from grapes, were studied regarding their physiological behavior (dry cell weight—DCW production, substrate uptake, and ethanol and glycerol biosynthesis) during fermentations of grape must, in some cases enriched with commercial glucose and fructose (initial total sugar concentration approximately 150 and 250 g/L, respectively). Myclobutanil (a chiral triazole fungicide broadly used as a protective agent of vine) was also added to the culture media at various concentrations in order to assess the ability of the yeasts to simultaneously perform alcoholic fermentations and detoxify the medium (i.e., to remove the fungicide). In the first set of experiments and for both tested strains, trials were carried out in either 250 mL or 2.0 L agitated shake flasks in either synthetic glucose-based experiments or grape musts. Since the results obtained in the trials where the cultures were placed in 2.0 L flasks with grape musts as substrates were superior in terms of both DCW and ethanol production, these experimental conditions were selected for the subsequent studies. Both strains showed high fermentative efficiency, producing high amounts of DCW (9.5–10.5 g/L) in parallel with high ethanol production, which in some cases achieved values very close to the maximum theoretical ethanol production yield (≈0.49 g of ethanol per g of sugar). When using grape must with initial total sugars at approximately 250 g/L (very high gravity fermentation media, close to winemaking conditions), significantly high ethanol quantities (i.e., ranging between 105 and 123 g/L) were produced. Myclobutanil addition slightly negatively affected sugar conversion into ethanol; however, in all cases, ethanol production was very satisfactory. A non-negligible myclobutanil removal during fermentation, which ranged between 5%–27%, as a result of the adsorptive or degradative capacity of the yeast was also reported. The presence of myclobutanil had no effect on DCW production and resulted in no significant differences in the biosynthesis of glycerol. Therefore, these newly isolated yeast strains could be excellent candidates for simultaneous high ethanol production and parallel pesticide removal in a general biorefinery concept demonstrating many environmental benefits.

Enhancement of glycerol production by UV-mutagenesis of the marine yeast Wickerhamomyces anomalus HH16: kinetics and optimization of the fermentation process.

The current study aims to enhance glycerol production using UV-mutagenesis of the marine yeast Wickerhamomyces anomalus HH16 isolated from marine sediment collected from South Sinai Governorate, Egypt. Besides optimization of the culture conditions and analyzing the kinetic parameters of growth and glycerol biosynthesis by the mutant strain were studied. The marine yeast isolate HH16 was selected as the front runner glycerol-producer among all tested isolates, with glycerol yield recorded as 66.55gl-1. The isolate was identified based on the phenotypic and genotypic characteristics of W. anomalus. The genotypic characterization based on the internal transcribed spacer (ITS) sequence was deposited in the GenBank database with the accession number MK182824. UV-mutagenesis of W. anomalus HH16 by its exposure to UV radiation (254nm, 200 mWcm-2) for 5min; increased its capability in the glycerol production rate with 16.97% (80.15gl-1). Based on the kinetic and Monod equations, the maximum specific growth rate (μ max) and maximum specific glycerol production rate (v max) by the mutant strain W. anomalus HH16MU5 were 0.21h-1 and 0.103gg-1, respectively. Optimization of the fermentation parameters such as nitrogen source, salinity and pH has been achieved. The maximum glycerol production 86.55gl-1 has been attained in a fermentation medium composed of 200gl-1 glucose, 1gl-1 peptone, 3gl-1 yeast extract, and 58.44gl-1 NaCl, this medium was adjusted at pH 8 and incubated for 3days at 30°C. Moreover, results indicated the ability of this yeast to produce glycerol (73.33gl-1) using a seawater based medium. These findings suggest the applicability of using the yeast isolate W. anomalus HH16MU5 as a potential producer of glycerol for industrial purposes.

A chromosome-level genome assembly reveals the genetic basis of cold tolerance in a notorious rice insect pest, Chilo suppressalis.

The rice stem borer, Chilo suppressalis, is one of the most damaging insect pests to rice production worldwide. Although C.suppressalis has been the focus of numerous studies examining cold tolerance and diapause, plant-insect interactions, pesticide targets and resistance, and the development of RNAi-mediated pest management, the absence of a high-quality genome has limited deeper insights. To address this limitation, we generated a draft C.suppressalis genome constructed from both Illumina and PacBio sequences. The assembled genome size was 824.35Mb with a contig N50 of 307kb and a scaffold N50 of 1.75Mb. Hi-C scaffolding assigned 99.2% of the bases to one of 29 chromosomes. Based on universal single-copy orthologues (BUSCO), the draft genome assembly was estimated to be 97% complete and is predicted to encompass 15,653 protein-coding genes. Cold tolerance is an extreme survival strategy found in animals. However, little is known regarding the genetic basis of the winter ecology of C.suppressalis. Here, we focused our orthologous analysis on those gene families associated with animal cold tolerance. Our finding provided the first genomic evidence revealing specific cold-tolerant strategies in C.suppressalis, including those involved in glucose-originated glycerol biosynthesis, triacylglycerol-originated glycerol biosynthesis, fatty acid synthesis and trehalose transport-intermediate cold tolerance. The high-quality C.suppressalis genome provides a valuable resource for research into a broad range of areas in molecular ecology, and subsequently benefits developing modern pest control strategies.

Metabolomics Profiling and AKR Characterization During Paurometabolous Development of Corythucha ciliata (Hemiptera: Tingidae).

The sycamore lace bug, Corythucha ciliata (Say) is an invasive pest infesting trees of the genus Platanus. Both adults and nymphs damage the foliage of sycamore trees. Nymphs cannot survive in low temperatures; however, the sycamore lace bug overwinters as adults. In this study, we analyzed the metabolite profiles of this pest to determine significantly regulated metabolites during paurometabolous development from nymphs to adults. The identification of metabolites is essential to convert analytical data into meaningful biological knowledge. A total of 62 metabolites were identified using GC-MS. Among them, 29 different metabolites showed differences in content among nymphs, adult females (AF), and adult males (AM). Five of the 29 metabolites, including caffeic acid, D-glucose, D-mannose, glycerol and aminooxyacetic acid, were significantly increased and nine of them were significantly decreased during the developmental stages from nymph to adult. In addition, we identified three novel aldo-keto reductase (AKR) genes that may play a significant role in the control of glycerol biosynthesis. Moreover, the characteristics and expression levels of these genes were analyzed. This study will provide us with the necessary information to improve our understanding of the changes in metabolites in C. ciliata during paurometabolous development.

1726. Candida albicans Virulence Genes Induced During Intra-abdominal Candidiasis (IAC) in the Absence of Antifungal Exposure Mediate Echinocandin Resistance

BackgroundWe developed and validated a mouse model of C. albicans IAC that mimics peritonitis and abscesses (IAA) in humans, and that is amenable to temporal–spatial transcriptional profiling and virulence studies.MethodsWe measured C. albicans SC5314 gene expression by RNA-Seq (RiboPure extraction; Illumina MiSeq) in triplicate during early peritonitis (within 30 minutes of infection), late peritonitis (24 hours) and IAA (48 hours). Differential expression was defined by ≥2-fold differences at false discovery rate ≤0.01.Results≥7 million C. albicans reads were detected in each experiment. 67% of C. albicans reads mapped to coding sequences, covering 93% of open reading frames. The 50 C. albicans genes most highly expressed during early peritonitis were associated with pH (including RIM101 and PHR1) and oxidative stress responses, and adhesion/hyphal growth (e.g., ALS3, HWP1, ECM331, SAP6). The corresponding 50 C. albicans late peritonitis genes were associated with neutrophil/macrophage responses and nutrient acquisition (glyoxylate cycle, fatty acid β-oxidation, iron homeostasis). Responses within IAA included DNA damage and iron metabolism, reflecting stress response and nutrient/metal limitation. The top 50 core gene responses for all stages were associated with adhesion, stress response, and glucose transport. Among the most up-regulated genes in late peritonitis and IAA compared with early peritonitis were those involved antifungal drug resistance (CDR family, MDR1, FLU1, and ERG family), although mice were not exposed to antifungals. Null and reconstitution mutants for genes involved in adhesion (ALS3), copper transport (CCC2), DNA (DDI1) and cell wall damage responses (DAP1 homologs), and glycerol biosynthesis (RHR2) were attenuated for virulence in temporal-spatial fashion during peritonitis and IAA, and/or hyper-susceptible to phagocytosis and echinocandins (table).Conclusion C. albicans relies upon diverse biologic processes to cause peritonitis and IAA. Multiple genes induced in response to stress during IAC mediate virulence, phagocyte, and echinocandin resistance. Therefore, pathogenic strategies used by C. albicans during IAC may lessen responses to echinocandin treatment, even in the absence of drug exposure or FKS mutations. Disclosures All authors: No reported disclosures.

BIOTECHNOLOGY OF ALCOHOLIC FERMENTATION WITH YEAST RECIRCULATION

In recent decades, there has been a tendency in the world to increase ethanol production significantly in order to solve energy problems, that is, to use it as a biofuel. The factors determining the production cost of targeted biotechnological products include the output of these products from the raw materials used. One of the modern and effective ways to intensify alcoholic fermentation and reduce the cost of fuel ethanol is yeast recirculation. The research objects were: raw material (sugarbeet molasses), molasses wort, yeast Saccharomyces cerevisiae of the strain M-5, fermented wash and its distillates. In the raw materials, intermediate products, and fermented wash, the techno-chemical parameters recommended by the current technology regulations for obtaining spirit from molasses have been determined. Acoholic fermentation was carried out in an industrial environment, in a battery of series-connected fermentors. Recirculation of yeast was carried out by separating it from the final stage of fermentation, concentrating it on the separator, and introducing it into the first fermentor. The experimental data obtained prove that for wort fermentation, it is effective to use yeast that recirculates in the anaerobic stage. It has been established that the alcohol-forming power of recycled yeast increases as the yeast adapts to the environment in which it has been staying for a long time. The yeast becomes more active biochemically, with more efficient metabolism. Its need for continuously cultured biomass is reduced, the share of aerobically assimiliated sugars decreases, and, consequently, the losses during yeast generation are fewer. At the same time, accelerating the initial period of anaerobic fermentation helps inhibit the biosynthesis of glycerol, the formation of which consumes the largest amount of sugar among all the secondary products. The parameters of molasses wort fermentation, with yeast biomass recirculating, have been determined in an industrial environment. It has been established that the alcohol output from the raw materials increases as the synthesis of secondary metabolic products weakens. The advantages of this fermentation method will be used in further studies, namely when fermenting molasses wort, with an increased concentration of dry matter, in order to reduce the specific heat energy consumption in production and to make it cheaper. The developed biotechnology of alcohol can be usefully employed to produce fuel ethanol, and increasing its production will contribute to Ukraine’s energy self-sufficience.

Ste20 and Cla4 modulate the expression of the glycerol biosynthesis enzyme Gpd1 by a novel MAPK-independent pathway

p21-activated kinases (PAKs) are important signalling molecules with a wide range of functions. In budding yeast, the main PAKs Ste20 and Cla4 regulate the response to hyperosmotic stress, which is an excellent model for the adaptation to changing environmental conditions. In this pathway, the only known function of Ste20 and Cla4 is the activation of a mitogen-activated protein kinase (MAPK) cascade through Ste11. This eventually leads to increased transcription of glycerol biosynthesis genes, the most important response to hyperosmotic shock. Here, we show that Ste20 and Cla4 not only stimulate transcription, they also bind to the glycerol biosynthesis enzymes Gpd1, Gpp1 and Gpp2. Protein levels of Gpd1, the enzyme that catalyzes the rate limiting step in glycerol synthesis, positively correlate with glucose availability. Using a chemical genetics approach, we find that simultaneous inactivation of STE20 and CLA4 reduces the glucose-induced increase of Gpd1 levels, whereas the deletion of either STE20 or CLA4 alone has no effect. This is also observed for the hyperosmotic stress-induced increase of Gpd1 levels. Importantly, under both conditions the deletion of STE11 has no effect on Gpd1 induction. These observations suggest that Ste20 and Cla4 not only have a role in the transcriptional regulation of GPD1 through Ste11. They also seem to modulate GPD1 expression at another level such as translation or protein degradation.

Dynamics of sugars, anaerobic metabolism enzymes and metabolites in apples stored under dynamic controlled atmosphere

Apple storage under extremely low oxygen concentrations, as in dynamic controlled atmosphere, changes both aerobic and anaerobic metabolism. In this work we evaluated the effects of controlled atmosphere (CA) and dynamic controlled atmosphere based on respiratory quotient (DCA – RQ), with RQ 1.3 and RQ 1.5, on the dynamics of pyruvic acid, sugars, anaerobic metabolites and enzymes involved in anaerobic metabolism of ‘Elstar’ and ‘Nicoter’ apples after harvest, 6 and 9 months of storage plus 7 days of shelf life. We also investigated the induction of sorbitol and glycerol biosynthesis, as a response to low oxygen stress in apples stored under DCA – RQ, protecting the cell membrane from leakage. Storage under CA and DCA – RQ had different dynamics of sugars accumulation from harvest up to 9 months of storage, especially for sorbitol, which accumulated more over the storage period when fruit are stored under DCA - RQ. Glycerol was not detected in any of the cultivars or storage conditions. Storage under DCA reduces the membrane permeability even with the accumulation of anaerobic metabolism compounds, like acetaldehyde and ethanol. Perhaps, this is a result of the higher sorbitol accumulation, which acts as osmolyte. For both cultivars, the storage under DCA resulted in an increase of PDC enzyme activity from harvest to 9 months of storage. The dynamics of anaerobic metabolism compounds were different for both cultivars: ‘Elstar’ apples showed an increase from harvest to 9 months storage, but ‘Nicoter’ had an increase until 6 months of storage and a sharp reduction until 9 months of storage. The regulation of anaerobic metabolism is performed by PDC enzyme activity, with little influence of ADH enzyme activity, when apples are stored under DCA – RQ. ‘Nicoter’ apples are much more sensitive to low oxygen stress conditions as compared to ‘Elstar’.

Endocannabinoid contributions to alcohol habits and motivation: Relevance to treatment.

Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a critical role in habit learning and in ethanol self-administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride-induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2-AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol-motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder.

Osmolyte accumulation regulates the SUMOylation and inclusion dynamics of the prionogenic Cyc8-Tup1 transcription corepressor.

Environmental stressors can severely perturb cellular homeostasis and compromise viability. To cope with environmental stressors, eukaryotes have developed distinct signaling programs that allow for adaptation during different stress conditions. These programs often require a host of post-translational modifications that alter proteins to elicit appropriate cellular responses. One crucial protein modifier during stress is the small ubiquitin-like modifier SUMO. In many cases, however, the functions of stress dependent protein SUMOylation remain unclear. Previously, we showed that the conserved Saccharomyces cerevisiae Cyc8-Tup1 transcriptional corepressor complex undergoes transient hyperosmotic stress-induced SUMOylation and inclusion formation, which are important for appropriate regulation of hyperosmotic-stress genes. Here, we show the osmostress-responsive MAP kinase Hog1 regulates Cyc8 SUMOylation and inclusion formation via its role in the transcriptional activation of glycerol biosynthesis genes. Mutations that ablate Cyc8 SUMOylation can partially rescue the osmosensitivity of hog1Δ cells, and this is facilitated by inappropriate derepression of glycerol-biosynthesis genes. Furthermore, cells specifically unable to synthesize the osmolyte glycerol cause transient Cyc8 SUMOylation and inclusions to persist, indicating a regulatory role for glycerol to reestablish the basal state of Cyc8 following adaptation to hyperosmotic stress. These observations unveil a novel intersection between phosphorylation and SUMOylation networks, which are critical for shifting gene expression and metabolic programs during stress adaptation.

Linking gene expression and oenological traits: Comparison between Torulaspora delbrueckii and Saccharomyces cerevisiae strains

Wine fermentations typically involve the yeast Saccharomyces cerevisiae. However, many other yeast species participate to the fermentation process, some with interesting oenological traits. In this study the species Torulaspora delbrueckii, used occasionally in mixed or sequential fermentation with S. cerevisiae to improve wine sensory profile, was investigated to understand the physiological differences between the two. Next generation sequencing was used to characterize the transcriptome of T. delbrueckii and highlight the different genomic response of these yeasts during growth under wine-like conditions. Of particular interest were the basic differences in the glucose fermentation pathway and the formation of aromatic and flavour compounds such as glycerol, esters and acetic acid. Paralog genes were missing in glycolysis and glycerol biosynthesis in T. delbrueckii. Results indicate the tendency of T. delbrueckii to produce less acetic acid relied on a higher expression of alcoholic fermentation related genes, whereas acetate esters were influenced by the absence of esterases, ATF1–2. Additionally, in the Δbap2 S. cerevisiae strain, the final concentration of short branched chain ethyl esters (SBCEEs) was related to branched chain amino acid (BCAA) uptake. In conclusion, different adaption strategies are apparent for T. delbrueckii and S. cerevisiae yeasts, an understanding of which will allow winemakers to make better use of such microbial tools to achieve a desired wine sensory outcome.

Novel Histidine Kinase Gene HisK2301 from Rhodosporidium kratochvilovae Contributes to Cold Adaption by Promoting Biosynthesis of Polyunsaturated Fatty Acids and Glycerol.

Hybrid histidine kinase (HHKs) are widespread in fungi, but their roles in the regulation of fungal adaptation to environmental stresses remain largely unclear. To elucidate this, we cloned HisK2301 from Rhodosporidium kratochvilovae strain YM25235, characterized HisK2301 as a novel HHK, and further investigated the role of HisK2301 by overexpressing it in YM25235. Our results revealed that HisK2301 can promote adaptation of YM25235 to cold, osmotic, and salt stresses. During cold stress, HisK2301 significantly enhanced the biosynthesis of polyunsaturated fatty acids (PUFA) and intracellular glycerol. HisK2301 also augmented the expression levels of Δ12/Δ15 fatty acid desaturase ( RKD12) and glycerol-3-phosphate dehydrogenase1 ( GPD1), which are responsible for PUFA and glycerol biosynthesis, respectively. To conclude, our findings give the first insight into the defense and mechanisms of HisK2301 in fungi against cold stress and suggest the potential use of the novel cold-adapted HHK HisK2301 in industrial processes, such as large-scale production of PUFA.

Predicted Glycerol 3-Phosphate Dehydrogenase Homologs and the Glycerol Kinase GlcA Coordinately Adapt to Various Carbon Sources and Osmotic Stress in Aspergillus fumigatus.

Glycerol plays an important role in the adaptation of fungi to various microenvironments and stressors, including heat shock, anoxic conditions and osmotic stress. Glycerol 3-phosphate dehydrogenase (G3PDH) is able to catalyze dihydroxyacetone phosphate to glycerol 3-phosphate (G3P), which is subsequently dephosphorylated into glycerol. However, current knowledge about the functions of G3PDH homologs in glycerol biosynthesis in Aspergillus fumigatus is limited. Here, we show that the A. fumigatus G3PDH gene, gfdA, is crucial for normal colony growth in glucose media under both normoxic and hypoxic conditions. In addition, failure of the overexpression of the gfdA homolog, gfdB, to rescue the phenotype of a gfdA null mutant suggests that gfdA plays a predominant role in the synthesis of G3P and glycerol. However, in a wild-type background, overexpressing either gfdA or gfdB is able to significantly enhance biomass production of mycelia, suggesting that gfdA and gfdB have similar functions in promoting the use of glucose. Interestingly, overexpression of the gene encoding the predicted glycerol kinase, GlcA, which is capable of phosphorylating glycerol to form G3P, significantly rescues the growth defects of gfdA null mutants in glucose media, indicating that the growth defects of gfdA null mutants might be due to the absence of G3P rather than glycerol. Moreover, Western blotting analysis revealed that gfdA is inducibly expressed by osmotic mediators. However, in the absence of gfdA, osmotic stress can rescue colony growth defects and allow colonies to partially bypass the gfdA requirement in a high osmolarity glycerol pathway-dependent manner. Therefore, the findings of this study elucidate how saprophytic filamentous fungi have developed pathways distinct from those of budding yeasts to adapt to varied carbon sources and survive environmental stresses.

The enhancement of the oxidative pentose phosphate pathway maybe involved in resolving imbalance between photosystem I and II in Dunaliella salina

Dunaliella salina is a unicellular green alga extensively studied due to its capability of salt stress tolerance and high economic value for β-carotene production. Glycerol is synthesized to regulate intracellular osmotic pressure during salt stress in D. salina. In order to demonstrate the electron and reducing power distribution pattern in this process, photosynthetic performance and oxidative pentose phosphate pathway (OPPP) operation during salt stress were investigated. Glycerol was accumulated massively when D. salina was subjected to salt stress. Meanwhile, photosystem II (PSII) activity was increased while photosystem I (PSI) activity was decreased. This suggests that a small portion of the electrons generated in PSII were leaked and were transferred to generate reactive oxygen species (ROS), while most electrons subsequently used to PSI to generate ATP and NADPH. Furthermore, the OPPP was enhanced in order to provide reducing power and carbon skeleton for glycerol synthesis. In conclusion, during salt stress, the imbalance of photosynthetic electron distribution between PSII and PSI in D. salina resulted in a shortage of NADPH and ATP for glycerol biosynthesis, which also led the enhancement of the OPPP and ROS generation.