Valeria Mapelli

Metabolic footprinting in microbiology : methods and applications in functional genomics and biotechnology

Metabolomics embraces several strategies that aim to quantify cell metabolites in order to increase our understanding of how metabolite levels and interactions influence phenotypes. Metabolic footprinting represents a niche within metabolomics, because it focuses on the analysis of extracellular metabolites. Although metabolic footprinting represents only a fraction of the entire metabolome, it provides important information for functional genomics and strain characterization, and it can also provide scientists with a key understanding of cell communication mechanisms, metabolic engineering and industrial biotechnological processes. Due to the tight and convoluted relationship between intracellular metabolism and metabolic footprinting, metabolic footprinting can provide precious information about the intracellular metabolic status. Hereby, we state that integrative information from metabolic footprinting can assist in further interpretation of metabolic networks.

The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae

BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(±0.005) to 0.08(±0.002) and the anabolic reduction charge decreased from 0.46(±0.11) to 0.27(±0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(±0.002) to 0.85(±0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group “Cell rescue, defense and virulence” was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.

Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production

ABSTRACT Yeast has long been considered the microorganism of choice for second-generation bioethanol production due to its fermentative capacity and ethanol tolerance. However, tolerance toward inhibitors derived from lignocellulosic materials is still an issue. Flocculating yeast strains often perform relatively well in inhibitory media, but inhibitor tolerance has never been clearly linked to the actual flocculation ability per se. In this study, variants of the flocculation gene FLO1 were transformed into the genome of the nonflocculating laboratory yeast strain Saccharomyces cerevisiae CEN.PK 113-7D. Three mutants with distinct differences in flocculation properties were isolated and characterized. The degree of flocculation and hydrophobicity of the cells were correlated to the length of the gene variant. The effect of different strength of flocculation on the fermentation performance of the strains was studied in defined medium with or without fermentation inhibitors, as well as in media based on dilute acid spruce hydrolysate. Strong flocculation aided against the readily convertible inhibitor furfural but not against less convertible inhibitors such as carboxylic acids. During fermentation of dilute acid spruce hydrolysate, the most strongly flocculating mutant with dense cell flocs showed significantly faster sugar consumption. The modified strain with the weakest flocculation showed a hexose consumption profile similar to the untransformed strain. These findings may explain why flocculation has evolved as a stress response and can find application in fermentation-based biorefinery processes on lignocellulosic raw materials.

Metabolic and bioprocess engineering for production of selenized yeast with increased content of seleno-methylselenocysteine

Specific Se-metabolites have been recognized to be the main elements responsible for beneficial effects of Se-enriched diet, and Se-methylselenocysteine (SeMCys) is thought to be among the most effective ones. Here we show that an engineered Saccharomyces cerevisiae strain, expressing a codon optimized heterologous selenocysteine methyltransferase and endowed with high intracellular levels of S-adenosyl-methionine, was able to accumulate SeMCys at levels higher than commercial selenized yeasts. A fine tuned carbon- and sulfate-limited fed-batch bioprocess was crucial to achieve good yields of biomass and SeMCys. Through the coupling of metabolic and bioprocess engineering we achieved a ∼24-fold increase in SeMCys, compared to certified reference material of selenized yeast. In addition, we investigated the interplay between sulfur and selenium metabolism and the possibility that redox imbalance occurred along with intracellular accumulation of Se. Collectively, our data show how the combination of metabolic and bioprocess engineering can be used for the production of selenized yeast enriched with beneficial Se-metabolites.

The interplay between sulphur and selenium metabolism influences the intracellular redox balance in Saccharomyces cerevisiae

Selenium (Se) is an essential element for most eukaryotic organisms, including humans. The balance between Se toxicity and its beneficial effects is very delicate. It has been demonstrated that a diet enriched with Se has cancer prevention potential in humans. The most popular commercial Se supplementation is selenized yeast, which is produced in a fermentation process using an inorganic source of Se. Here, we show that the uptake of Se, Se toxic effects and intracellular Se-metabolite profile are largely influenced by the level of sulphur source supplied during the fermentation. A Yap1-dependent oxidative stress response is active when yeast actively metabolizes Se, and this response is linked to the generation of intracellular redox imbalance. The redox imbalance derives from a disproportionate ratio between the reduced and oxidized forms of glutathione and also from the influence of Se metabolism on the central carbon metabolism. The observed increase in glycerol production rate, concomitant with the inhibition of ethanol formation in the presence of Se, can be ascribed to the occurrence of redox imbalance that triggers glycerol biosynthesis to replenish the pool of NAD(+) .

Common and Distant Structural Characteristics of Feruloyl Esterase Families from Aspergillus oryzae

Background Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing attention on these enzymes, a novel descriptor based classification system has been proposed for FAEs resulting into 12 distinct families and pharmacophore models for three FAE sub-families have been developed. Methodology/Principal Findings The feruloylome of Aspergillus oryzae contains 13 predicted FAEs belonging to six sub-families based on our recently developed descriptor-based classification system. The three-dimensional structures of the 13 FAEs were modeled for structural analysis of the feruloylome. The three genes coding for three enzymes, viz., A.O.2, A.O.8 and A.O.10 from the feruloylome of A. oryzae, representing sub-families with unknown functional features, were heterologously expressed in Pichia pastoris, characterized for substrate specificity and structural characterization through CD spectroscopy. Common feature-based pharamacophore models were developed according to substrate specificity characteristics of the three enzymes. The active site residues were identified for the three expressed FAEs by determining the titration curves of amino acid residues as a function of the pH by applying molecular simulations. Conclusions/Significance Our findings on the structure-function relationships and substrate specificity of the FAEs of A. oryzae will be instrumental for further understanding of the FAE families in the novel classification system. The developed pharmacophore models could be applied for virtual screening of compound databases for short listing the putative substrates prior to docking studies or for post-processing docking results to remove false positives. Our study exemplifies how computational predictions can complement to the information obtained through experimental methods.

Enhancement of anaerobic lysine production in Corynebacterium glutamicum electrofermentations

It has been suggested that application of electric potential can affect lysine producing fermentations, although experimental evidence is lacking. To study this hypothesis we used the lysine producer Corynebacterium glutamicum ZW04, and we exposed it to 12 different conditions regarding anaerobic gas environment, applied electrode potential (cathodic, open circuit, anodic), redox mediator and nitrate presence. The gas environment was found to play a major role, with CO2 leading to double the lysine concentrations and yields when compared to N2. Electrode potentials also played a major role, with reductive conditions doubling the titers and increasing the yields of lysine up to 4 times. Addition of the redox mediator anthraquinone-2-sulfonate (AQ2S) under the presence of CO2 and reductive conditions led to additional doubling of the titers, although the yields were not altered considerably. This study demonstrates for the first time that cathodic electrode conditions combined with CO2 and AQ2S as a redox mediator can significantly improve both the yields and the titers of lysine production of a C. glutamicum lysine producing strain, reaching levels that have only been achieved under aerobic conditions.

The Presence of Pretreated Lignocellulosic Solids from Birch during Saccharomyces cerevisiae Fermentations Leads to Increased Tolerance to Inhibitors – A Proteomic Study of the Effects

The fermentation performance of Saccharomyces cerevisiae in the cellulose to ethanol conversion process is largely influenced by the components of pretreated biomass. The insoluble solids in pretreated biomass predominantly constitute cellulose, lignin, and -to a lesser extent- hemicellulose. It is important to understand the effects of water-insoluble solids (WIS) on yeast cell physiology and metabolism for the overall process optimization. In the presence of synthetic lignocellulosic inhibitors, we observed a reduced lag phase and enhanced volumetric ethanol productivity by S. cerevisiae CEN.PK 113-7D when the minimal medium was supplemented with WIS of pretreated birch or spruce and glucose as the carbon source. To investigate the underlying molecular reasons for the effects of WIS, we studied the response of WIS at the proteome level in yeast cells in the presence of acetic acid as an inhibitor. Comparisons were made with cells grown in the presence of acetic acid but without WIS in the medium. Altogether, 729 proteins were detected and quantified, of which 246 proteins were significantly up-regulated and 274 proteins were significantly down-regulated with a fold change≥1.2 in the presence of WIS compared to absence of WIS. The cells in the presence of WIS up-regulated several proteins related to cell wall, glycolysis, electron transport chain, oxidative stress response, oxygen and radical detoxification and unfolded protein response; and down-regulated most proteins related to biosynthetic pathways including amino acid, purine, isoprenoid biosynthesis, aminoacyl-tRNA synthetases and pentose phosphate pathway. Overall, the identified differentially regulated proteins may indicate that the likelihood of increased ATP generation in the presence of WIS was used to defend against acetic acid stress at the expense of reduced biomass formation. Although, comparative proteomics of cells with and without WIS in the acetic acid containing medium revealed numerous changes, a direct effect of WIS on cellular physiology remains to be investigated.

Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage.

Cathodes enhance Corynebacterium glutamicum growth with nitrate and promote acetate and formate production

The industrially important Corynebacterium glutamicum can only incompletely reduce nitrate into nitrite which then accumulates and inhibits growth. Herein we report that cathodes can resolve this problem and enhance glucose fermentation and growth by promoting nitrite reduction. Cell growth was inhibited at relatively high potentials but was significant when potentials were more reductive (-1.20V with anthraquinone-2-sulfonate as redox mediator or -1.25V vs. Ag/AgCl). Under these conditions, glucose was consumed up to 6 times faster and acetate was produced at up to 11 times higher yields (up to 1.1mol/mol-glucose). Acetate concentrations are the highest reported so far for C. glutamicum under anaerobic conditions, reaching values up to 5.3±0.3g/L. Herein we also demonstrate for the first time formate production (up to 3.4±0.3g/L) by C. glutamicum under strongly reducing conditions, and we attribute this to a possible mechanism of CO2 bioreduction that was electrochemically triggered.