High-level Production of Isoleucine and Fusel alcohol by expression of the Feedback Inhibition-insensitive Threonine deaminase in Saccharomyces cerevisiae

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2-3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Isolation and characterization of yeast for the production of rice wine with low fusel alcohol content

Fusel alcohols (FAs) are a type of flavor compound found in rice wine. An overly high FA content not only leads to spicy, bitter, and astringent taste but also has side effects. Therefore, screening for yeast that produce low FA contents has attracted much attention. Thirty-two yeast strains were isolated from fermenting material during Luzhou-flavor liquor production in this study. Strain YB-12 was selected as a suitable candidate for rice wine production. The strain was identified as a member of the genus Meyerozyma based on phylogenetic analysis using 26S rDNA gene sequences. The ability of strain YB-12 to produce ethanol was similar to that of Saccharomyces cerevisiae NRRL Y-567, while isobutanol and isoamyl alcohol production was only 53.96% and 50.23%, respectively, of that of NRRL Y-567. The FA yield of rice wine produced with strain YB-12 was reduced to 51.85% in a 20 L fermenter. These results demonstrate that strain YB-12 presents promising characteristics for use in the production of rice wine with a potentially low content of FAs.

Value-Added Products from Ethanol Fermentation—A Review

Global demand for renewable and sustainable energy is increasing, and one of the most common biofuels is ethanol. Most ethanol is produced by Saccharomyces cerevisiae (yeast) fermentation of either crops rich in sucrose (e.g., sugar cane and sugar beet) or starch-rich crops (e.g., corn and starchy grains). Ethanol produced from these sources is termed a first-generation biofuel. Yeast fermentation can yield a range of additional valuable co-products that accumulate during primary fermentation (e.g., protein concentrates, water soluble metabolites, fusel alcohols, and industrial enzymes). Distillers’ solubles is a liquid co-product that can be used in animal feed or as a resource for recovery of valuable materials. In some processes it is preferred that this fraction is modified by a second fermentation with another fermentation organism (e.g., lactic acid bacteria). Such two stage fermentations can produce valuable compounds, such as 1,3-propanediol, organic acids, and bacteriocins. The use of lactic acid bacteria can also lead to the aggregation of stillage proteins and enable protein aggregation into concentrates. Once concentrated, the protein has utility as a high-protein feed ingredient. After separation of protein concentrates the remaining solution is a potential source of several known small molecules. The purpose of this review is to provide policy makers, bioethanol producers, and researchers insight into additional added-value products that can be recovered from ethanol beers. Novel products may be isolated during or after distillation. The ability to isolate and purify these compounds can provide substantial additional revenue for biofuel manufacturers through the development of marketable co-products.

Norwegian Kveik brewing yeasts are adapted to higher temperatures and produce fewer off-flavours under heat stress than commercial Saccharomyces cerevisiae American Ale yeast

Norwegian kveik are a recently described family of domesticated Saccharomyces cerevisiae brewing yeasts used by farmhouse brewers in western Norway for generations to produce traditional Norwegian farmhouse ale. Kveik ale yeasts have been domesticated by farmhouse brewers through serial repitching of the yeast in warm wort (>30°C) punctuated by long periods of dry storage. Kveik yeasts are alcohol tolerant, flocculant, capable of utilizing maltose/maltotriose, phenolic off flavour negative, and exhibit elevated thermotolerance when compared to other modern brewer's yeasts belonging to the 'Beer 1' clade. However, the optimal fermentation and growth temperatures (Topt) for kveik ale yeasts and the influence of fermentation temperature of the production of flavour-active metabolites like fusel alcohols and sulfur compounds (H2S, SO2) are not known. Here we show that kveik ale yeasts have an elevated optimal fermentation temperature (Topt) when compared to commercial American Ale yeast (SafAle™ US-05) and that they produce fewer off-flavours at high temperatures (>30°C) when compared to commercial American Ale yeasts. The tested kveik yeasts show significantly higher maximum fermentation rates than American Ale yeast not only at elevated temperatures (>30°C), but also at 'typical' ale fermentation temperatures (20°C-25°C). Finally, we demonstrate that kveik ale yeasts are heterogeneous in their Topt and that they attenuate standard wort robustly above their Topt unlike our control American Ale yeast which showed very poor apparent attenuation in our standard wort at temperatures >> Topt. Our results provide further support that kveik yeasts may possess favourable fermentation kinetics and sensory properties compared to American Ale yeasts. The observations here provide a roadmap for brewers to fine tune their commercial fermentations using kveik ale yeasts for optimal performance and/or flavour impact.

Characterization of a Spirit Beverage Produced with Strawberry Tree (Arbutus unedo L.) Fruit and Aged with Oak Wood at Laboratorial Scale

Arbutus unedo spirit is a valuable product in Mediterranean countries. This spirit is usually marketed in Portugal without wood ageing. This work aims to characterize the ageing effect on the Arbutus unedo spirit, for three and six months with oak wood (Quercus robur L.) submitted to different toasting levels, based on its chemical composition and its sensory properties. For this purpose, several parameters were analysed: acidity, pH, dry extract, and volatile compounds (methanol, acetaldehyde, ethyl acetate and fusel alcohols). The volatile compounds were identified by GC-MS and quantified by GC-FID. Sensory analysis was performed by a trained panel, who have profiled this beverage, as well as the changes acquired during ageing. Spectroscopic techniques, namely FTIR–ATR, were applied to discriminate the different beverages produced. The results highlighted an increase in Arbutus unedo spirit’s quality with the wood contact, mainly based on the sensory attributes. Additionally, they showed that the best beverages were produced using oak wood with medium toasting levels during three months of ageing.

Nitrogenous Compound Utilization and Production of Volatile Organic Compounds among Commercial Wine Yeasts Highlight Strain-Specific Metabolic Diversity

ABSTRACTStrain and environmental nutrient concentrations can affect the production of sensory impact compounds during yeast fermentation. Despite reports on the impact of nutrient conditions on kinetics of cellular growth, it is uncertain to what extent nitrogen utilization by commercial Saccharomyces cerevisiae wine strains affects the production of volatile organic (aroma) compounds (VOCs). Here we ask whether i) consumption of amino acids contribute to VOCs (fusel alcohols, acetate esters, and fatty acid esters) in commercial S. cerevisiae yeast strains, ii) there is inter-strain variation in VOC production, and iii) there is a correlation between the production of aroma compounds and nitrogen utilization. We analyzed the consumption of nutrients as well as the production of major VOCs during fermentation of a chemically defined grape juice medium with four commercial S. cerevisiae yeast strains: Elixir, Opale, R2, and Uvaferm. The production of VOCs was variable among the strains where Uvaferm correlated with ethyl acetate and ethyl hexanoate production, R2 negatively correlated with the acetate esters, and Opale positively correlated with fusel alcohols. The four strains’ total biomass formation was similar, pointing to metabolic differences in the utilization of nutrients to form secondary metabolites such as VOCs. To understand the strain-dependent differences in VOC production, partial least-squares linear regression coupled with genome-scale metabolic modeling was performed with the objective to correlate nitrogen utilization with fermentation biomass and volatile formation. Total aroma production was found to be a strong function of nitrogen utilization (R2 = 0.87). We found that glycine, tyrosine, leucine, and lysine utilization were positively correlated with fusel alcohols and acetate esters concentrations e.g., 2-phenyl acetate during wine fermentation. Parsimonious flux balance analysis and flux enrichment analysis confirmed the usage of these nitrogen utilization pathways based on the strains’ VOC production phenotype.IMPORTANCESaccharomyces cerevisiae is widely used in grape juice fermentation to produce wines. Along with the genetic background, the nitrogen in the environment in which S. cerevisiae grows impacts its regulation of metabolism. Also, commercial S. cerevisiae strains exhibit immense diversity in their formation of aromas, and a desirable aroma bouquet is an essential characteristic for wines. Since nitrogen affects aroma formation in wines, it is essential to know the extent of this connection and how it leads to strain-dependent aroma profiles in wines. We evaluated the differences in the production of key aroma compounds among four commercial wine strains. Moreover, we analyzed the role of nitrogen utilization on the formation of various aroma compounds. This work illustrates the unique aroma producing differences among industrial yeast strains and suggests more intricate, nitrogen associated routes influencing those aroma producing differences.

Autochthonous Yeast from Pork and Game Meat Fermented Sausages for Application in Meat Protection and Aroma Developing

The wild yeast community was studied in fermented sausages from pork and game meat (deer and wild boar) during the maturation process from different curing rooms. Although the biotechnological importance of yeasts in the maturation process of pork sausages is known, there is a lack of information for sausage maturation involving game meat. A total of 123 yeasts were isolated and, by amplifying and sequencing of the ITS region, were classified in 14 species. Debaryomyces hansenii, Kazachstania servazzii, and Wickerhamomyces anomalus were isolated in both pork and game samples. The PCR-RAPD technique differentiated between 26 and 18 strains from pork and game meat sausages, respectively. The physicochemical parameters and their relationship with the yeast community were also studied. The antioxidant and anti-lipid peroxidation capability were analyzed and the 70% and 50% of the tested strains showed these abilities, respectively. Moreover, the biocontrol capability against mycotoxigenic molds was found in 19 strains, but better results were observed in game meat yeasts. On the other hand, almost 30% of strains produce a pleasant olfactory aroma, and volatile compounds associated with the yeast pathway metabolic during the maturation process have been characterized such as esters, aldehydes, fusel alcohols, etc. This study has allowed a better understanding of the biodiversity of this type of food, as well as selecting potential yeast strains for their future use as starters.

The Use of Non-Saccharomyces Yeast and Enzymes in Beer Production

The objective of this paper was to test the potential of selected non-Saccharomyces strains for beer production, by using Saccharomyces cerevisiae as a control sample. For some of variants brewing enzymes were added to wort to increase the content of fermentable sugars. The non-Saccharomyces yeasts differed in the fermentation process rate. The basic beer physiochemical parameters were assessed, including: alcohol content, extract, free amino nitrogen, sugars, acidity, colour, and the profile of volatile compounds and metal ions. The use of enzymes caused an increase in alcohol and fusel alcohols concentration in beers obtained. Total acidity, free amine nitrogen content, colour and sugar content indicated that the tested non-Saccharomyces yeast allowed obtaining beers with the proper analytical parameters.