The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

Enhanced fermentative capacity of yeasts engineered in storage carbohydrate metabolism

During yeast biomass production, cells are grown through several batch and fed‐batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench‐top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic capacity of yeast which can be an interesting application for the food industry. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:20–24, 2015     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Gaining insight into the response logic of Saccharomyces cerevisiae to heat shock by combining expression profiles with metabolic pathways

Extensive alteration of gene expression and metabolic remodeling enable the budding yeast Saccharomyces cerevisiae to ensure cellular homeostasis and adaptation to heat shock. The response logic of the cells to heat shock is still not entirely clear. In this study, we combined the expression profiles with metabolic pathways to investigate the logical relations between heat shock response metabolic pathways. The results showed that the heat-stressed S. cerevisiae cell accumulated trehalose and glycogen, which protect cellular proteins against denaturation, and modulate its phospholipid structure to sustain stability of the cell wall. The TCA cycle was enhanced, and the heat shock-induced turnover of amino acids and nucleotides served to meet the extra energy requirement due to heat-induced protein metabolism and modification. The enhanced respiration led to oxidative stress, and subsequently induced the aldehyde detoxification system. These results indicated that new insight into the response logic of S. cerevisiae to heat shock can be gained by integrating expression profiles and the logical relations between heat shock response metabolic pathways.     

A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions

High gravity (HG) or very high gravity (VHG) brewing has become popular in modern breweries due to its economic and product quality advantages. However, there are the negative impacts such as the fermentation performance of brewer??s yeast in HG or VHG wort, which are closely related to changes in cell physiological activity. In the present study, 3 kinds of worts, with different gravities, were used to examine the systematic effects on fermentation performance and physiological activity of lager yeast FBY009505 (Saccharomyces pastorianus) and ale yeast FBY0099 (Saccharomyces cerevisiae), as well as the resulting beer flavor. Results showed that the responses of FBY009505 and FBY0099 to the HG or VHG worts were similar. The specific fermentation rate and viability of cropped yeast of FBY009505 and FBY0099 were decreased with increasing wort gravity. The increased wort gravity resulted in the increase of energy charge and the decrease of ??-glucosides transport rate and glycolytic enzyme activities. Moreover, the environmental stresses in the HG or VHG wort showed a higher inhibitory activity against ??-glucoside transport than glycolytic enzymes. The content of intracellular trehalose and glycerol of FBY009505 and FBY0099 increased with the increase in wort gravity. The results from this study provided a potential means to systematically understand the physiology of brewer??s yeast under HG or VHG conditions.     

Comparative analysis of glycogen and trehalose accumulation in methylotrophic and nonmethylotrophic yeasts

Trehalose and glycogen accumulate in certain yeast species when they are exposed to unfavorable growth conditions. Accumulations of these reserve carbohydrates in yeasts provide resistance to stress conditions. The results of this study indicate that certain Pichia species do not accumulate high levels of glycogen and trehalose under normal growth conditions. However, depending on the Pichia species, both saccharides accumulate at high levels when the Pichia cells are exposed to unfavorable or stress-inducing growth conditions. Growth in glycerol or methanol medium mostly led to trehalose accumulation in Pichia species tested in this study. It was shown that the metabolic pathways for glycogen and trehalose biosynthesis are present in Pichia species. However, it appears that the biosynthesis of trehalose and glycogen may be regulated in different manners in Pichia species than in the yeast S. cerevisiae. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 6, pp. 737–741. The text was submitted by the author in English.     

Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil

In this work, 74 Saccharomyces cerevisiae strains isolated from cachaça fermentation of six different geographic regions in Brazil were characterized by mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) and by their ability to grow on stress conditions occurring during the cachaça fermentation process. Cachaça S. cerevisiae strains showed high mtDNA-RFLP polymorphism with the occurrence of 32 different molecular patterns. The S. cerevisiae strains presenting prevalent mtDNA were able to grow better in the stress conditions than strains represented by infrequent patterns. The principal coordinate analysis on 17 stress conditions revealed that the major source of growth variation were high ethanol concentrations and low temperatures. These results indicate that the stress conditions occurring in the fermentation process influence the prevalence of the most adapted S. cerevisiae strains in each distillery. The physiological tests used in our study can be used as a criterion for rapidly selecting autochthonous yeast strains for further purposes such as the selection of fermentative starters of S. cerevisiae strains.     

Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene

This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.     

Data integration uncovers the metabolic bases of phenotypic variation in yeast

The relationship between different levels of integration is a key feature for understanding the genotype-phenotype map. Here, we describe a novel method of integrated data analysis that incorporates protein abundance data into constraint-based modeling to elucidate the biological mechanisms underlying phenotypic variation. Specifically, we studied yeast genetic diversity at three levels of phenotypic complexity in a population of yeast obtained by pairwise crosses of eleven strains belonging to two species, Saccharomyces cerevisiae and S. uvarum. The data included protein abundances, integrated traits (life-history/fermentation) and computational estimates of metabolic fluxes. Results highlighted that the negative correlation between production traits such as population carrying capacity (K) and traits associated with growth and fermentation rates (J_max) is explained by a differential usage of energy production pathways: a high K was associated with high TCA fluxes, while a high J_max was associated with high glycolytic fluxes. Enrichment analysis of protein sets confirmed our results.This powerful approach allowed us to identify the molecular and metabolic bases of integrated trait variation, and therefore has a broad applicability domain.     

Enological properties in wild and commercial Saccharomyces cerevisiae yeasts: relationship with competition during alcoholic fermentation

Commercial yeasts are widely used in winemaking to carry out the alcoholic fermentation; nevertheless, some wild strains may compete with them and even dominate the process. In this research, 21 prevailing wild strains isolated from inoculated musts were chosen in order to study the competition between them and commercial yeasts. Some biotechnological properties which could enhance their performance during the process were also studied, such as vitality, killer factor, resistance to high concentrations of sugar, ethanol and SO₂ or trehalose and glycogen cell content. All yeasts, both commercial and wild strains, showed resistance to the killer toxin and they were all able to growth under adverse conditions. However, vitality and carbohydrate content were strain dependent in around 70% of the cases; the wild strain had a higher vitality and accumulated less trehalose than its commercial counterpart.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Fumaric Acid Production in Saccharomyces cerevisiae by In Silico Aided Metabolic Engineering

Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L^–1 without any apparent change in growth in fed-batch culture. FT-IR and ¹H and ¹³C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L^–1 FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L^–1 FA in batch culture when the SFC1 gene encoding a succinate–fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering.     

Changes in the Pi uptake and polyP accumulation in Saccharomyces cerevisiae strains deficient in the synthesis of trehalose and/or glycerol

The intracellular level of free inorganic orthophosphate (P_i) in yeast cells generally depends on the P_i uptake capacity, energy state of the cells in respect to the activity of the membrane-associated ATPases and on the activity of metabolic pathways involved in the production of glycerol and trehalose. Batch fermentation was performed to investigate the carbon substrate consumption, the P_i uptake capacity and product formation by four Saccharomyces cerevisiae strains differing in their ability to produce glycerol and/or trehalose. The consumption of P_i in mutant strains with a lack of the synthesis of the trehalose and/or glycerol exceeded the level for a wild type strain about two times. Maximum intracellular polyP content (29.9 mg/g DW) was shown for tps1Δ gpd1Δ mutant. In this study we showed that the P_i uptake and polyP accumulation level were closely connected with the changes in the synthesis of trehalose and glycerol.     

Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism.     

Carbohydrate Metabolism During Ascospore Development in Yeast

Carbohydrate metabolism, under sporulation conditions, was compared in sporulating and non-sporulating diploids of Saccharomyces cerevisiae. Total carbohydrate was fractionated into trehalose, glycogen, mannan, and an alkali-insoluble fraction composed of glucan and insoluble glycogen. The behavior of three fractions was essentially the same in both sporulating and non-sporulating strains; trehalose, mannan, and the insoluble fraction were all synthesized to about the same extent regardless of a strain's ability to undergo meiosis or sporulation. In contrast, aspects of soluble glycogen metabolism depended on sporulation. Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores. We also determined the carbohydrate composition of spores isolated from mature asci. Spores contained all components present in vegetative cells, but in different proportions. In cells, the most abundant carbohydrate was mannan, followed by glycogen, then trehalose, and finally the alkali-insoluble fraction; in spores, trehalose was most abundant, followed by the alkali-insoluble fraction, glycogen, and mannan in that order.     

Behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel and performing alcoholic fermentation

Summary The behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel was studied during their continuous function in an ethanol-producing reactor. Polymerization destroys 40% to 80% of the cells, depending on their physiological state. A three day adaptation phase is required before ethanol production stabilizes and this phase corresponds to an increase in cell concentration in the gels and to protein synthesis. The amounts of DNA, glucan, glycogen and trehalose are different in entrapped and free cells. Microscopic observation shows that 75% to 85% of the cells lose their integrity and that the remainder appear to multiply normally. Within a gel particle, both viability and fermentation activity are heterogeneous. A high percentage of cells have low viability and low fermentation activity. A proportion of cells remains capable of forming colonies and these cells have higher fermentation activity and are preferentially localized at the surface of gel particles.