Corrigendum: Investigating the Effect of Selected Non-Saccharomyces Species on Wine Ecosystem Function and Major Volatiles.

[This corrects the article DOI: 10.3389/fbioe.2018.00169.].

Investigating the Effect of Selected Non-Saccharomyces Species on Wine Ecosystem Function and Major Volatiles.

Natural alcoholic fermentation is initiated by a diverse population of several non-Saccharomyces yeast species. However, most of the species progressively die off, leaving only a few strongly fermentative species, mainly Saccharomyces cerevisiae. The relative performance of each yeast species is dependent on its fermentation capacity, initial cell density, ecological interactions as well as tolerance to environmental factors. However, the fundamental rules underlying the working of the wine ecosystem are not fully understood. Here we use variation in cell density as a tool to evaluate the impact of individual non-Saccharomyces wine yeast species on fermentation kinetics and population dynamics of a multi-species yeast consortium in synthetic grape juice fermentation. Furthermore, the impact of individual species on aromatic properties of wine was investigated, using Gas Chromatography-Flame Ionization Detector. Fermentation kinetics was affected by the inoculation treatment. The results show that some non-Saccharomyces species support or inhibit the growth of other non-Saccharomyces species in the multi-species consortium. Overall, the fermentation inoculated with a high cell density of Starmerella bacillaris displayed the fastest fermentation kinetics while fermentation inoculated with Hanseniaspora vineae showed the slowest kinetics. The production of major volatiles was strongly affected by the treatments, and the aromatic signature could in some cases be linked to specific non-Saccharomyces species. In particular, Wickerhamomyces anomalus at high cell density contributed to elevated levels of 2-Phenylethan-1-ol whereas Starm. bacillaris at high cell density resulted in the high production of 2-methylpropanoic acid and 3-Hydroxybutanone. The data revealed possible direct and indirect influences of individual non-Saccharomyces species within a complex consortium, on wine chemical composition.

Cloning the putative gene of vinyl phenol reductase of Dekkera bruxellensis in Saccharomyces cerevisiae.

Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for "Brett" sensory modification of wine, has been recently recognized to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismutase and as a NADH-dependent reductase. The present study aimed for providing a certain identification of the enzyme by cloning the VPR gene in S. cerevisiae, a species not producing ethyl phenols. Transformed clones of S. cerevisiae resulted capable of expressing a biologically active form of the heterologous protein, proving its role in the conversion of 4-vinyl guaiacol to 4-ethyl guaiacol. A VPR specific protein activity of 9 ± 0.6 mU/mg was found in crude extracts of S. cerevisiae recombinant strain. This result was confirmed in activity trials carried out with the protein purified from transformant cells of S. cerevisiae by a his-tag purification approach; in particular, VPR-enriched fractions showed a specific activity of 1.83 ± 0.03 U/mg at pH 6.0. Furthermore, in agreement with literature, the purified protein behaves like a SOD, with a calculated specific activity of approximatively 3.41 U/mg. The comparative genetic analysis of the partial VPR gene sequences from 17 different D. bruxellesis strains suggested that the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not justify the well described strain-dependent character in producing volatile phenols of this species. Actually, no correlation exists between genotype membership of the analysed strains and their capability to release off-flavours. This work adds valuable knowledge to the study of D. bruxellensis wine spoilage and prepare the ground for interesting future industrial applications.

Effects of Oxygen Availability on Acetic Acid Tolerance and Intracellular pH in Dekkera bruxellensis.

UNLABELLED: The yeast Dekkera bruxellensis, associated with wine and beer production, has recently received attention, because its high ethanol and acid tolerance enables it to compete with Saccharomyces cerevisiae in distilleries that produce fuel ethanol. We investigated how different cultivation conditions affect the acetic acid tolerance of D. bruxellensis We analyzed the ability of two strains (CBS 98 and CBS 4482) exhibiting different degrees of tolerance to grow in the presence of acetic acid under aerobic and oxygen-limited conditions. We found that the concomitant presence of acetic acid and oxygen had a negative effect on D. bruxellensis growth. In contrast, incubation under oxygen-limited conditions resulted in reproducible growth kinetics that exhibited a shorter adaptive phase and higher growth rates than those with cultivation under aerobic conditions. This positive effect was more pronounced in CBS 98, the more-sensitive strain. Cultivation of CBS 98 cells under oxygen-limited conditions improved their ability to restore their intracellular pH upon acetic acid exposure and to reduce the oxidative damage to intracellular macromolecules caused by the presence of acetic acid. This study reveals an important role of oxidative stress in acetic acid tolerance in D. bruxellensis, indicating that reduced oxygen availability can protect against the damage caused by the presence of acetic acid. This aspect is important for optimizing industrial processes performed in the presence of acetic acid. IMPORTANCE: This study reveals an important role of oxidative stress in acetic acid tolerance in D. bruxellensis, indicating that reduced oxygen availability can have a protective role against the damage caused by the presence of acetic acid. This aspect is important for the optimization of industrial processes performed in the presence of acetic acid.

Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma.

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; in contrast to most other known ketoreductases, KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates. As a result, a new useful catalyst was identified, enabling the enantioselective preparation of different aromatic alcohols and hydroxyketones.

An efficient continuous flow process for the synthesis of a non-conventional mixture of fructooligosaccharides.

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity. Moreover, the stability of this production system was ascertained by up to 7 days of continuous working. These results suggest the suitability of the proposed method for a large-scale production of the desired FOS mixture, in view of a foreseeable use as a novel prebiotic preparation.

Cold exposure affects carbohydrates and lipid metabolism, and induces Hog1p phosphorylation in Dekkera bruxellensis strain CBS 2499.

Dekkera bruxellensis is a yeast known to affect the quality of wine and beer. This species, due to its high ethanol and acid tolerance, has been reported also to compete with Saccharomyces cerevisiae in distilleries producing fuel ethanol. In order to understand how this species responds when exposed to low temperatures, some mechanisms like synthesis and accumulation of intracellular metabolites, changes in lipid composition and activation of the HOG-MAPK pathway were investigated in the genome sequenced strain CBS 2499. We show that cold stress caused intracellular accumulation of glycogen, but did not induce accumulation of trehalose and glycerol. The cellular fatty acid composition changed after the temperature downshift, and a significant increase of palmitoleic acid was observed. RT-PCR analysis revealed that OLE1 encoding for Δ9-fatty acid desaturase was up-regulated, whereas TPS1 and INO1 didn't show changes in their expression. In D. bruxellensis Hog1p was activated by phosphorylation, as described in S. cerevisiae, highlighting a conserved role of the HOG-MAP kinase signaling pathway in cold stress response.