Impact of Starmerella bacillaris and Zygosaccharomyces bailii on ethanol reduction and Saccharomyces cerevisiae metabolism during mixed wine fermentations

The bulk of grape juice fermentation is carried out by the yeast Saccharomyces cerevisiae, but non-Saccharomyces yeasts can modulate many sensorial aspects of the final products in ways not well understood. In this study, some of such non-conventional yeasts were screened as mixed starter cultures in a fermentation defined medium in both simultaneous and sequential inoculations. One strain of Starmerella bacillaris and another of Zygosaccharomyces bailii were chosen by their distinct phenotypic footprint and their ability to reduce ethanol levels at the end of fermentation, particularly during simultaneous vinification. S. bacillaris losses viability strongly at the end of mixed fermentation, while Z. bailii remains viable until the end of vinification. Interestingly, for most non-Saccharomyces yeasts, simultaneous inoculation helps for survival at the end of fermentation compared to sequential inoculation. S. cerevisiae viability was unchanged by the presence of the either yeast. Characterization of both strains indicates that S. bacillaris behavior is overall more different from S. cerevisiae than Z. bailii. S. bacillaris has a less strict glucose repression mechanism and molecular markers like catabolite repression kinase Snf1 is quite different in size. Besides, S. cerevisiae transcriptome changes to a bigger degree in the presence of S. bacillaris than when inoculated with Z. bailii. S. bacillaris induces the translation machinery and repress vesicular transport. Both non-Saccharomyces yeast induce S. cerevisiae glycolytic genes, and that may be related to ethanol lowering, but there are specific aspects of carbon-related mechanisms between strains: Z. bailii presence increases the stress-related polysaccharides trehalose and glycogen while S. bacillaris induces gluconeogenesis genes.

Mechanisms of Metabolic Adaptation in Wine Yeasts: Role of Gln3 Transcription Factor

Wine strains of Saccharomyces cerevisiae have to adapt their metabolism to the changing conditions during their biotechnological use, from the aerobic growth in sucrose-rich molasses for biomass propagation to the anaerobic fermentation of monosaccharides of grape juice during winemaking. Yeast have molecular mechanisms that favor the use of preferred carbon and nitrogen sources to achieve such adaptation. By using specific inhibitors, it was determined that commercial strains offer a wide variety of glucose repression profiles. Transcription factor Gln3 has been involved in glucose and nitrogen repression. Deletion of GLN3 in two commercial wine strains produced different mutant phenotypes and only one of them displayed higher glucose repression and was unable to grow using a respiratory carbon source. Therefore, the role of this transcription factor contributes to the variety of phenotypic behaviors seen in wine strains. This variability is also reflected in the impact of GLN3 deletion in fermentation, although the mutants are always more tolerant to inhibition of the nutrient signaling complex TORC1 by rapamycin, both in laboratory medium and in grape juice fermentation. Therefore, most aspects of nitrogen catabolite repression controlled by TORC1 are conserved in winemaking conditions.

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Cellular heterogeneity and MTH1 play key roles in galactose mediated signaling of the GAL switch to utilize the disaccharide melibiose

Yeast metabolizes the disaccharide melibiose by hydrolyzing it into equimolar concentrations of glucose and galactose by MEL1-encoded α-galactosidase. Galactose metabolizing genes (including MEL1) are induced by galactose and repressed by glucose, which are the products of melibiose hydrolysis. Therefore, how melibiose catabolization and utilization take place by circumventing the glucose repression is an enigma. Other than the galactose metabolizing genes MTH1, a negative regulator of glucose signal pathway has Gal4p binding sites and is induced by galactose and repressed by high glucose concentration. But, at low or no glucose MTH1 along with its paralogue STD1 represses hexose transporters, that are involved in glucose transport. This sort of tuning of glucose and galactose regulation motivated us to delineate the role of MTH1 as a regulator of MEL1 expression and melibiose utilization. The deletion mutant of MTH1 shows growth defect on melibiose and this growth defect is enhanced upon the deletion of both MTH1 and its paralogue STD1. Microscopy and flowcytometry analysis, suggest, that even though MEL1 and GAL1 promoter are under Gal4p and Gal80p regulation, upon deletion of MTH1 it hampers only MEL1 expression, but not the GAL1 gene expression. By using 2-Deoxy galactose toxicity assay, we observed phenotypic heterogeneity in cells grown on melibiose i.e. after cleaving of melibiose a fraction of cell population utilizes glucose and another fraction utilizes galactose and coexist together. Understanding GAL/MEL gene expression patterns in melibiose will have great implication to understand various other complex sugar utilizations, tunable gene expressions and complex feedback gene regulations.

Improvement of Enantiomeric l-Lactic Acid Production from Mixed Hexose-Pentose Sugars by Coculture of Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9

Among 39 pentose-utilizing lactic acid bacteria (LAB) selected from acid-forming bacteria from the midgut of Eri silkworm, the isolate WX1 was selected with the highest capability to produce optically pure l-lactic acid (l-LA) from glucose, xylose and arabinose with furfural-tolerant properties. The isolate WX1 was identified as Enterococcus mundtii based on 16S rDNA sequence analysis. The conversion yields of l-LA from glucose and xylose by E. mundtii WX1 were 0.97 and 0.68 g/g substrate, respectively. Furthermore, l-LA production by E. mundtii WX1 in various glucose-xylose mixtures indicated glucose repression effect on xylose consumption. The coculture of E. mundtii WX1 and Lactobacillus rhamnosus SCJ9, a homofermentative LAB capable of producing l-LA from glucose clearly showed an improvement of l-LA production from 30 g/L total glucose-xylose (6:4). The results from Plackett–Burman design (PBD) indicated that Tween 80, MnSO4 and yeast extract (YE) were three medium components that significantly influenced (p < 0.05) l-LA production using the coculture strategy in the presence of 2 g/L furfural. Optimal concentrations of these variables revealed by central composite design (CCD) and response surface methodology (RSM) were 20.61 g/L YE, 1.44 g/L Tween 80 and 1.27 g/L MnSO4. Based on the optimized medium with 30 g/L total glucose-xylose (6:4), the maximum experimental l-LA value of 23.59 g/L reflecting 0.76 g/g substrate were achieved from 48 h fermentation at 37 °C. l-LA produced by coculture cultivated under standard MRS medium and new optimized conditions were 1.28 and 1.53 times higher than that obtained from single culture by E. mundtii WX1, respectively. This study provides the foundations for practical applications of coculture in bioconversion of lignocellulose particularly glucose-xylose-rich corn stover to l-LA.

Role of Elm1, Tos3, and Sak1 Protein Kinases in the Maltose Metabolism of Baker’s Yeast

Glucose repression is a key regulatory system controlling the metabolism of non-glucose carbon source in yeast. Glucose represses the utilization of maltose, the most abundant fermentable sugar in lean dough and wort, thereby negatively affecting the fermentation efficiency and product quality of pasta products and beer. In this study, the focus was on the role of three kinases, Elm1, Tos3, and Sak1, in the maltose metabolism of baker’s yeast in lean dough. The results suggested that the three kinases played different roles in the regulation of the maltose metabolism of baker’s yeast with differential regulations on MAL genes. Elm1 was necessary for the maltose metabolism of baker’s yeast in maltose and maltose-glucose, and the overexpression of ELM1 could enhance the maltose metabolism and lean dough fermentation ability by upregulating the transcription of MALx1 (x is the locus) in maltose and maltose-glucose and MALx2 in maltose. The native level of TOS3 and SAK1 was essential for yeast cells to adapt glucose repression, but the overexpression of TOS3 and SAK1 alone repressed the expression of MALx1 in maltose-glucose and MALx2 in maltose. Moreover, the three kinases might regulate the maltose metabolism via the Snf1-parallel pathways with a carbon source-dependent manner. These results, for the first time, suggested that Elm1, rather than Tos3 and Sak1, might be the dominant regulator in the maltose metabolism of baker’s yeast. These findings provided knowledge about the glucose repression of maltose and gave a new perspective for breeding industrial yeasts with rapid maltose metabolism.

Candida albicans Hexokinase 2 Challenges the Saccharomyces cerevisiae Moonlight Protein Model

Survival of the pathogenic yeast Candida albicans depends upon assimilation of fermentable and non-fermentable carbon sources detected in host microenvironments. Among the various carbon sources encountered in a human body, glucose is the primary source of energy. Its effective detection, metabolism and prioritization via glucose repression are primordial for the metabolic adaptation of the pathogen. In C. albicans, glucose phosphorylation is mainly performed by the hexokinase 2 (CaHxk2). In addition, in the presence of glucose, CaHxK2 migrates in the nucleus and contributes to the glucose repression signaling pathway. Based on the known dual function of the Saccharomyces cerevisiae hexokinase 2 (ScHxk2), we intended to explore the impact of both enzymatic and regulatory functions of CaHxk2 on virulence, using a site-directed mutagenesis approach. We show that the conserved aspartate residue at position 210, implicated in the interaction with glucose, is essential for enzymatic and glucose repression functions but also for filamentation and virulence in macrophages. Point mutations and deletion into the N-terminal region known to specifically affect glucose repression in ScHxk2 proved to be ineffective in CaHxk2. These results clearly show that enzymatic and regulatory functions of the hexokinase 2 cannot be unlinked in C. albicans.

A novel yeast hybrid modeling framework integrating Boolean and enzyme-constrained networks enables exploration of the interplay between signaling and metabolism

The interplay between nutrient-induced signaling and metabolism plays an important role in maintaining homeostasis and its malfunction has been implicated in many different human diseases such as obesity, type 2 diabetes, cancer, and neurological disorders. Therefore, unraveling the role of nutrients as signaling molecules and metabolites together with their interconnectivity may provide a deeper understanding of how these conditions occur. Both signaling and metabolism have been extensively studied using various systems biology approaches. However, they are mainly studied individually and in addition, current models lack both the complexity of the dynamics and the effects of the crosstalk in the signaling system. To gain a better understanding of the interconnectivity between nutrient signaling and metabolism in yeast cells, we developed a hybrid model, combining a Boolean module, describing the main pathways of glucose and nitrogen signaling, and an enzyme-constrained model accounting for the central carbon metabolism of Saccharomyces cerevisiae, using a regulatory network as a link. The resulting hybrid model was able to capture a diverse utalization of isoenzymes and to our knowledge outperforms constraint-based models in the prediction of individual enzymes for both respiratory and mixed metabolism. The model showed that during fermentation, enzyme utilization has a major contribution in governing protein allocation, while in low glucose conditions robustness and control are prioritized. In addition, the model was capable of reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression, as well as regulatory effects associated with lifespan increase during caloric restriction. Overall, we show that our hybrid model provides a comprehensive framework for the study of the non-trivial effects of the interplay between signaling and metabolism, suggesting connections between the Snf1 signaling pathways and processes that have been related to chronological lifespan of yeast cells.

Auxin-mediated protein depletion for metabolic engineering in terpene-producing yeast

In metabolic engineering, loss-of-function experiments are used to understand and optimise metabolism. A conditional gene inactivation tool is required when gene deletion is lethal or detrimental to growth. Here, we exploit auxin-inducible protein degradation as a metabolic engineering approach in yeast. We demonstrate its effectiveness using terpenoid production. First, we target an essential prenyl-pyrophosphate metabolism protein, farnesyl pyrophosphate synthase (Erg20p). Degradation successfully redirects metabolic flux toward monoterpene (C10) production. Second, depleting hexokinase-2, a key protein in glucose signalling transduction, lifts glucose repression and boosts production of sesquiterpene (C15) nerolidol to 3.5 g L−1 in flask cultivation. Third, depleting acetyl-CoA carboxylase (Acc1p), another essential protein, delivers growth arrest without diminishing production capacity in nerolidol-producing yeast, providing a strategy to decouple growth and production. These studies demonstrate auxin-mediated protein degradation as an advanced tool for metabolic engineering. It also has potential for broader metabolic perturbation studies to better understand metabolism.

Acid pH Strategy Adaptation through NRG1 in Ustilago maydis

The role of the Ustilago maydis putative homolog of the transcriptional repressor ScNRG1, previously described in Saccharomyces cerevisiae, Candida albicans and Cryptococcus neoformans, was analyzed by means of its mutation. In S. cerevisiae this gene regulates a set of stress-responsive genes, and in C. neoformans it is involved in pathogenesis. It was observed that the U. maydisNRG1 gene regulates several aspects of the cell response to acid pH, such as the production of mannosyl-erythritol lipids, inhibition of the expression of the siderophore cluster genes, filamentous growth, virulence and oxidative stress. A comparison of the gene expression pattern of the wild type strain versus the nrg1 mutant strain of the fungus, through RNA Seq analyses, showed that this transcriptional factor alters the expression of 368 genes when growing at acid pH (205 up-regulated, 163 down-regulated). The most relevant genes affected by NRG1 were those previously reported as the key ones for particular cellular stress responses, such as HOG1 for osmotic stress and RIM101 for alkaline pH. Four of the seven genes included WCO1 codifying PAS domain ( These has been shown as the key structural motif involved in protein-protein interactions of the circadian clock, and it is also a common motif found in signaling proteins, where it functions as a signaling sensor) domains sensors of blue light, two of the three previously reported to encode opsins, one vacuolar and non-pH-responsive, and another one whose role in the acid pH response was already known. It appears that all these light-reactive cell components are possibly involved in membrane potential equilibrium and as virulence sensors. Among previously described specific functions of this transcriptional regulator, it was found to be involved in glucose repression, metabolic adaptation to adverse conditions, cellular transport, cell rescue, defense and interaction with an acidic pH environment.