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Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency. Results: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique. Conclusions: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

(2S)-eriodictyol (ERD) is a flavonoid widely found in citrus fruits, vegetables, and important medicinal plants with neuroprotective, cardioprotective, antidiabetic, and anti-obesity effects. However, the microbial synthesis of ERD is limited by complex metabolic pathways and often results in a low production performance. Here, we engineered Saccharomyces cerevisiae by fine-tuning the metabolism of the ERD synthesis pathway. The results showed that the ERD titer was effectively increased, and the intermediate metabolites levels were reduced. First, we successfully reconstructed the de novo synthesis pathway of p-coumaric acid in S. cerevisiae and fine-tuned the metabolic pathway using promoter engineering and terminator engineering for the high-level production of (2S)-naringenin. Subsequently, the synthesis of ERD was achieved by introducing the ThF3′H gene from Tricyrtis hirta. Finally, by multiplying the copy number of the ThF3′H gene, the production of ERD was further increased, reaching 132.08 mg L−1. Our work emphasizes the importance of regulating the metabolic balance to produce natural products in microbial cell factories. [ABSTRACT FROM AUTHOR]

As one of the microorganism commonly used in industrial production, Saccharomyces cerevisiae is of low pH resistance and strong stress resistance. The use of microorganisms to produce bio-based products has become an important development direction of green biomanufacturing. Isobutanol is a kind of branched-chain alcohol, which has a good application prospect in the chemical industry, energy, and other fields. As a biofuel, the synthesized isobutanol from S. cerevisiae is a sustainable, renewable, economical and environment-friendly approach to meet the needs of human development. The construction of an efficient isobutanol synthesizing S. cerevisiae cell factory may enable the green manufacturing of isobutanol as a biofuel. However, the ability of S. cerevisiae to synthesize isobutanol has limited the industrial development of isobutanol, the rational metabolic regulation strategy combined with synthetic biology is one of the effective methods to improve the ability of microbial isobutanol synthesis. This review summarized the latest developments in the regulating strategies of isobutanol biosynthesis in S. cerevisiae, including promoting isobutanol synthesis pathways, blocking competitive pathways, balancing cofactors, relocating synthetic pathways, regulating transcription factors, and improving microbial isobutanol tolerance. These strategies have significantly improved the conversion and yield of isobutanol in engineering yeast, but there are still many deficiencies in the industrial production of isobutanol. In order to promote the industrial production of isobutanol, the current bottlenecks and their solutions for isobutanol production in S. cerevisiae were discussed, aiming to provide valuable information for the green manufacturing of isobutanol. [ABSTRACT FROM AUTHOR]

Astaxanthin is a valuable orange-red carotenoid with wide applications in agriculture, food, cosmetics, pharmaceuticals and nutraceuticals areas. At present, the biological synthesis of astaxanthin mainly relies on Haematococcus pluvialis and Xanthophyllomyces dendrorhous. With the rapid development of synthetic biology, more recombinant microbial hosts have been genetically constructed for astaxanthin production including Escherichia coli , Saccharomyces cerevisiae and Yarrowia lipolytica. As multiple genes (15) were involved in the astaxanthin synthesis, it is particularly important to adopt different strategies to balance the metabolic flow towards the astaxanthin synthesis. Furthermore, astaxanthin is a fat-soluble compound stored intracellularly, hence efficient extraction methods are also essential for the economical production of astaxanthin. Several efficient and green extraction methods of astaxanthin have been reported in recent years, including the superfluid extraction, ionic liquid extraction and microwave-assisted extraction. Accordingly, this review will comprehensively introduce the advances on the astaxanthin production and extraction by using different microbial hosts and strategies to improve the astaxanthin synthesis and extraction efficiency. [ABSTRACT FROM AUTHOR]

Owing to the pharmaceutical value of retinal (vitamin A aldehyde), there is an increasing demand for the microbial synthesis of bio-based retinal. However, its microbial production is hindered by endogenous and redundant conversion of retinal to retinol and retinoic acid, which represents a major bottleneck for efficient retinal synthesis. In this study, we found that a combined inactivation of a functionally redundant subset of four alcohol dehydrogenases (Adh6, Adh7, Sfa1 and Gre2) and an aldehyde dehydrogenase (Hfd1) in Saccharomyces cerevisiae rendered the yeast with the ability to accumulate retinal. Stepwise optimization of β-carotene synthesis in the budding yeast harboring chromosomally integrated crtE/YB/I genes from Xanthophyllomyces dendrorhous and β-carotene 15,15′-monooxygenase (BCMO) from the marine bacterium 66A03 allowed us to produce 69.13 ± 5.82 mg L−1 retinal (>99% purity) in shake-flasks. Taken together, we have built a S. cerevisiae-based platform for retinal synthesis, which will pave the way for the future industrial development of bio-based retinal from renewable feedstocks. [ABSTRACT FROM AUTHOR]

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation. • A malonyl-CoA pathway was constructed with mutant Ehd3 and Acc1. • The catalytic activity of Ehd3 was significantly influenced with E124 site. • A fluorescence screening method was built to screen high-copy integration strains. • Pathway and fermentation optimization improved malonate titer by 14-fold. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle finishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-flow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × 108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1–8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9–11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No differences in pH, d-, l-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment effects were observed on NH3–N concentration; however, efficiency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3–N concentration was reduced to approximately 2 mg/dL as the challenge progressed. [ABSTRACT FROM AUTHOR]

Context: Dietary supplementation especially feed residues improve by yeast affected rumen fermentation. Aims: The aim of the present experiment was to determine the nutritive value, fermentation efficiency and rumen ecology of yeast-fermented cassava pulp, under the use of two levels of protein in concentrate mixtures, in beef cattle. Methods: Four beef cattle, 2–3 years of age, were randomly assigned to the following treatments according to a 2 × 2 factorial arrangement in a 4 × 4 Latin square design: cassava pulp fermented either with baker's yeast or LDD 6 (Factor A), with 16% or 18% CP in concentrate (Factor B). Key results: CP digestibility and blood urea nitrogen were higher in animals fed both baker's yeast-fermented and LDD 6-fermented cassava pulp with 18% CP than in those fed baker's yeast-fermented cassava pulp with 16% CP and LDD 6-fermented cassava pulp with 16% CP. The highest total volatile fatty acid (TVFA) and butyrate (C4) concentrations were in animals fed baker's yeast-fermented cassava pulp with 16% CP. Moreover, CH4 production was at its lowest when animals were fed with baker's yeast-fermented cassava pulp together with a concentrate with 16% CP. Animal's fed baker's yeast-fermented and LDD 6-fermented cassava pulp with 18% CP had higher concentrations of purine derivatives, higher microbial nitrogen supply and efficiency of microbial protein synthesis than did those fed either baker's yeast-fermented or LDD 6-fermented cassava pulp with 16% CP. In addition, protozoa population in animals fed baker's yeast-fermented cassava pulp was lower than in those fed LDD 6-fermented cassava pulp, and baker's yeast-fermented cassava pulp with 16% CP had the lowest protozoa population. However, F. succinogenes was the highest in animals fed yeast-fermented and LDD 6-fermented cassava pulp with 18% CP. Conclusions: It is concluded that yeast-fermented cassava pulp can be nutritionally improved, especially when used in combination with a concentrate of 16% CP to achieve the highest level of TVFA and C3 and to reduce rumen protozoal population and CH4 production. Implications: The present study suggests that feeding animals with yeast-fermented cassava pulp with a concentrate of 16% CP could improve feed quality and, hence, beef animal performance in Thailand. So as to compare cassava pulp in two forms, namely, fermented with baker's yeast or the yeast from LDD 6, under the use of two levels of protein in concentrate mixtures (16% and 18% crude protein, CP), four beef cattle were randomly assigned to treatments according to a 2 × 2 factorial arrangement in a 4 × 4 Latin square design. In conclusion, yeast-fermented cassava pulp with a concentrate of 16% CP resulted in the highest propionate production and a reduction of protozoal population and, hence, methane production in the rumen. [ABSTRACT FROM AUTHOR]

The researchers of this study investigated the biosynthesis of squalene by the yeast S. cerevisiae VGSH-2 through the activity of squalene epoxidase, which is a key enzyme in the conversion of squalene to ergosterol. It has been established that under aerobic conditions the antimycotic drug terbinafine promotes the switching of ergosterol formation to squalene synthesis. This switch occurs through specific inhibition of the squalene epoxidase of the yeast S. cerevisiae VGSH-2, thus increasing the biosynthetic ability of the yeast towards squalene. According to the results of this study, the optimal concentration of terbinofine in the nutrient medium was 0.3 µmol/cm³. This concentration led to a 5-fold decrease in squalene epoxidase activity and a 7-8 times increase in squalene synthesis. The results obtained can be used to develop a competitive technology for the industrial production of squalene by microbial synthesis. [ABSTRACT FROM AUTHOR]

Microbial production of oleochemicals from renewable feedstocks remains an attractive route to produce high-energy density, liquid transportation fuels and high-value chemical products. Metabolic engineering strategies have been applied to demonstrate production of a wide range of oleochemicals, including free fatty acids, fatty alcohols, esters, olefins, alkanes, ketones, and polyesters in both bacteria and yeast. The majority of these demonstrations synthesized products containing long-chain fatty acids. These successes motivated additional effort to produce analogous molecules comprised of medium-chain fatty acids, molecules that are less common in natural oils and therefore of higher commercial value. Substantial progress has been made towards producing a subset of these chemicals, but significant work remains for most. The other primary challenge to producing oleochemicals in microbes is improving the performance, in terms of yield, rate, and titer, of biocatalysts such that economic large-scale processes are feasible. Common metabolic engineering strategies include blocking pathways that compete with synthesis of oleochemical building blocks and/or consume products, pulling flux through pathways by removing regulatory signals, pushing flux into biosynthesis by overexpressing rate-limiting enzymes, and engineering cells to tolerate the presence of oleochemical products. In this review, we describe the basic fundamentals of oleochemical synthesis and summarize advances since 2013 towards improving performance of heterotrophic microbial cell factories. • Review of advances in microbial oleochemical production since 2013. • Overview of the structure and enzymology of oleochemical biosynthesis. • Summary of metabolic engineering strategies for improving oleochemical production. • Commentary on the state of oleochemical synthesis. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effect of adding selected amino acids (glutamate, proline, lysine and valine) to brewing wort on the generation of aroma active compounds along with the transcriptional profiling of genes involved in nitrogen regulation. The results showed that changes in the amino acid levels had no significant impact on the general course of fermentation except for the addition of lysine, which prolonged the period that lager yeast was in suspension. Flavour profile of lager were significantly affected by valine addition, which improved the degree of fermentation and alcohol concentration. Changes in the amount of isoamyl alcohol caused by varied amino acid conditions were particularly important as it fluctuated across the flavour threshold. Amino acid supplementation resulted in a reduction in yeast total amino acid utilisation except for valine. Furthermore, we found that nitrogen regulation genes were differentially expressed according to the amino acid added and stage of yeast growth, demonstrating that divergent mechanisms and pathways are involved in transcriptional regulation down‐regulating activators or up‐regulating repressors of nitrogen catabolite repression pathway at the mid‐exponential stage. An enexpected expression of genes related to SPS sensing system and the TOR pathway was found during the late exponential stage. Overall, our results indicate that, during lager yeast fermentation, the production of aroma active compounds and nitrogen regulation can be significantly affected by changing wort amino acid composition. © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

A statistical model was developed to describe the fermentation process using Saccharomyces cerevisiae ATCC 9763 in the production of black rice (cv. Poireton) wine from Manipur, India. Fermentation process parameters were optimised by response surface methodology and experiments were conducted according to a three‐level and three‐variable central composite rotatable design. The combination of pH, temperature and time was the most promising for achieving a high ethanol yield. With the use of the developed quadratic model equation, alcohol production (11.1%, v/v) was obtained in a batch fermentation process at the optimum operating condition of pH (6.96), temperature (26.2°C) and incubation time (74 h). GC–MS analysis of the wine revealed volatile aromatic compounds, namely 1‐methyl‐2‐pyrrolidine ethanol, propionic acid, 3‐mercapto and ethyl ester. © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

This study sought to select novel Saccharomyces cerevisiae strains not previously used in the production of beer. Twelve strains isolated from grape must, bakery, wine and apple stillage were compared through laboratory scale fermentation. Yeast from the grape must exhibited a superior fermentative capability (comparable with that of two commercial strains). Some of the strains produced appreciable concentrations of esters and higher alcohols, suggesting their potential as novel yeasts for beer characterised by distinct flavours. The most suitable strain for beer production, based on fermentative ability and volatile profile, was a baker's yeast which was subject to pilot plant scale‐up. The standard quality attributes, amino acids, volatile and sensory profiles were monitored during the primary fermentation and after bottle conditioning. These were aligned with those of a standard ale, suggesting that the selected yeast strain could be used for beer production. Interestingly, some esters were above the sensory threshold. © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

2-Phenylethanol (2-PE) is a kind of advanced aromatic alcohol with rose fragrance, which is wildly used for the deployment of flavors and fragrances. Microbial transformation is the most feasible method for the production of natural 2-PE. But a bottleneck problem is the toxicity of 2-PE on the cells. The molecular mechanisms of the toxic effect of 2-PE to Saccharomyces cerevisiae are not well studied. In this study, we analyzed the transcriptomes of S. cerevisiae in the media with and without 2-PE, respectively, using Illumina RNA-Seq technology. We identified 580 differentially expressed genes between S. cerevisiae in two different treatments. GO and KEGG enrichment analyses of these genes suggested that most genes encoding mitochondrial proteins, cytoplasmic, and plasma membrane proteins were significantly up-regulated, whereas the enzymes related to amino acid metabolism were down-regulated. These results indicated that 2-PE suppressed the synthesis of plasma membrane proteins, which suppressed the transport of nutrients required for growth. The findings in this study will provide insight into the inhibitory mechanism of 2-PE to yeast and other microbes. [ABSTRACT FROM AUTHOR]

In this study effects of commercial yeast preparations on the aromatic profile of Sauvignon Blanc varietal wine were investigated. Grape juice was divided to 7 experimental variants and fermented spontaneously and using 6 commercial strains of Saccharomyces cerevisiae. In final wine samples, essential analytical parameters and selected aromatic compounds were analysed. The highest content of esters was found out in samples fermented by spontaneous micro-flora; in this case, concentrations of ethyl hexanoate, ethyl octanoate and ethyl decanoate were 682 µg / L, 735 µg / L and 162 µg / L, respectively. The highest content of acetates was recorded in samples fermented by yeast Vulcaferm Sauvignon; concentrations of isoamyl acetate, 2-phenylethyl acetate and isobutyl acetate were 7.8 mg / L, 244 µg / L and 137 µg / L, respectively. Yeast strain suitable for cold fermentation (Oenoferm Fredo) produced high amounts of ethyl esters and acetates. As far as the sensory evaluation was concerned, the best rating got the sample fermented by these yeasts; it showed a high degree of smell and flavour cleanness as well as a very good overall harmony. [ABSTRACT FROM AUTHOR]

Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels. Up to now, this valuable terpene is commercially unavailable since there is no applicable manufacturing process. Microbial synthesis can be a promising route for sabinene production. In this review, we summarize knowledge about the metabolic pathway and key enzymes for sabinene biosynthesis. Recent advances that have been made in production of sabinene by microbial fermentation are highlighted. In these studies, researchers have identified the general synthetic pathway of sabinene from simple intermediate metabolites. Sabinene synthases of different origins were also cloned and characterized. Additionally, heterologous systems of the model microbes Escherichia coli and Saccharomyces cerevisiae were constructed to produce sabinene. This review also suggests new directions and attempts to gain some insights for achieving an industrial level production of sabinene. The combination of traditional molecular biology with new genome and proteome analysis tools will provide a better view of sabinene biosynthesis and a greater potential of microbial production. [ABSTRACT FROM AUTHOR]

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, P TDH3 , P FBA1 , and P TDH1 were favorable for high expression, and P SED1 , P HXT7 , P PDC1 , P TEF1 , P TPI1 , and P PGK1 were acceptable for medium–high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. P TEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium–high expression in glucose media. P ZWF1 and P SOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. P ALD3 and P TKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation. [ABSTRACT FROM AUTHOR]

Background: High temperature inhibits cell growth and ethanol fermentation of Saccharomyces cerevisiae. As a complex phenotype, thermotolerance usually involves synergistic actions of many genes, thereby being difficult to engineer. The overexpression of either endogenous or exogenous stress-related transcription factor genes in yeasts was found to be able to improve relevant stress tolerance of the hosts. Results: To increase ethanol yield of high-temperature fermentation, we constructed a series of strains of S. cerevisiae by expressing 8 transcription factor genes from S. cerevisiae and 7 transcription factor genes from thermotolerant K. marxianus in S. cerevisiae. The results of growth curve measurements and spotting test show that KmHsf1 and KmMsn2 can enhance cell growth of S. cerevisiae at 40-42 °C. According to the results of batch fermentation at 43 °C with an initial glucose concentration of 104.8 g/l, the fermentation broths of KmHSF1 and KmMSN2-expressing strains could reach final ethanol concentrations of 27.2 ± 1.4 and 27.6 ± 1.2 g/l, respectively, while the control strain just produced 18.9 ± 0.3 g/l ethanol. Transcriptomic analysis found that the expression of KmHSF1 and KmMSN2 resulted in 55 (including 31 up-regulated and 24 down-regulated) and 50 (including 32 up-regulated and 18 down-regulated) genes with different expression levels, respectively (padj < 0.05). The results of transcriptomic analysis also reveal that KmHsf1 might increase ethanol production by regulating genes related to transporter activity to limit excessive ATP consumption and promote the uptake of glucose; while KmMsn2 might promote ethanol fermentation by regulating genes associated with glucose metabolic process and glycolysis/gluconeogenesis. In addition, KmMsn2 might also help to cope with high temperature by regulating genes associated with lipid metabolism to change the membrane fluidity. Conclusions: The transcription factors KmHsf1 and KmMsn2 of thermotolerant K. marxianus can promote both cell growth and ethanol fermentation of S. cerevisiae at high temperatures. Different mechanisms of KmHsf1 and KmMsn2 in promoting high-temperature ethanol fermentation of S. cerevisiae were revealed by transcriptomic analysis. [ABSTRACT FROM AUTHOR]

Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the P GAL1 -controlled ISPS by overexpression of GAL 4 . Meanwhile, the native GAL1 /7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0 mg/L to 23.6 mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4 -overexpressing strain YXM29 achieved 50.2 mg/L of isoprene in sealed vials, and the isoprene production reached 640 mg/L and 3.7 g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals. [ABSTRACT FROM AUTHOR]

[Display omitted] • Various hemoglobins and myoglobins were synthetized in S. cerevisiae. • Subcellular localization of all enzymes involved in heme synthesis were confirmed. • The spatial barrier was removed by reconstructing heme synthetic pathway. • Enhancing precursor supply and assembling key enzymes to increase heme supply. • The biochemical properties of synthesized hemoglobins and myoglobins were detected. Hemoglobin (Hb) and myoglobin (Mb) are kinds of heme-binding proteins that play crucial physiological roles in different organisms. With rapid application development in food processing and biocatalysis, the requirement of biosynthetic Hb and Mb is increasing. However, the production of Hb and Mb is limited by the lower expressional level of globins and insufficient or improper heme supply. After selecting an inducible strategy for the expression of globins, removing the spatial barrier during heme synthesis, increasing the synthesis of 5-aminolevulinate and moderately enhancing heme synthetic rate-limiting steps, the microbial synthesis of bovine and porcine Hb was firstly achieved. Furthermore, an engineered Saccharomyces cerevisiae obtained a higher titer of soybean (108.2 ± 3.5 mg/L) and clover (13.7 ± 0.5 mg/L) Hb and bovine (68.9 ± 1.6 mg/L) and porcine (85.9 ± 5.0 mg/L) Mb. Therefore, this systematic engineering strategy will be useful to produce other hemoproteins or hemoenzymes with high activities. [ABSTRACT FROM AUTHOR]

Separate hydrolysis fermentation (SHF) and simultaneous saccharification fermentation (SSF) processes were studied for bioethanol production from microalgal biomass. SSF was selected as an efficient process to enhance the bioethanol yield through repeated-batches using immobilized yeast cells. Combined sonication and enzymatic hydrolysis of Chlamydomonas mexicana generated 10.5 and 8.48 g/L of ethanol in SSF and SHF, respectively. Yeast utilized maximum portion of total reducing sugar (TRS) reaching a consumption efficiency of 91–98%. A bioethanol yield of 0.5 g/g (88.2% of theoretical yield) and volumetric productivity of 0.22 g/L/h was obtained after 48 h of SSF. Immobilized yeast cells enabled repetitive production of ethanol for 7 cycles displaying a fermentation efficiency up to 79% for five consecutive cycles. The maximum ethanol production was 9.7 g/L in 2nd–4th cycles. A total energy recovery of 85.81% was achieved from microalgal biomass in the form of bioethanol. Repeated-batch SSF demonstrated the possibility of cost-effective bioethanol production. [ABSTRACT FROM AUTHOR]

The efficient fermentation of hydrolyzed sugars from lignocellulosic biomass feedstock to ethanol remains a complex multi-parametric problem. Thus, in the present study, an advanced structured dynamic model for the simulation of the fermentative ethanol production from hydrolysate sugars is developed. The model is combined with a statistical experimental design to determine an optimal operating strategy that maximizes ethanol production and serves for the systematic evaluation of critical process variables. In particular, the effects of various operating conditions and feeding strategies on the dynamic behavior of batch and fed-batch fermentation processes are explored. The deviation from the desired product or the metabolic inhibition of ethanol production are related with the applied environmental conditions and substrate and product inhibition phenomena. The operating strategy, designed with the assistance of the mathematical tools proposed in this study, includes an exponential addition policy of substrate. This strategy is experimentally proved to enhance the final product concentration, raising the ethanol productivity to 2.27 g L −1 h −1 and the ethanol yield to 53.5% of the maximum theoretical value. Moreover, the simulated strategies were in excellent agreement with the experimental results obtained from the real process using low and high glucose initial concentration, under batch and fed-batch conditions, in both flask- and bioreactor-scale cultivations, proving the model's predictive and optimization capabilities. Further improvement of process performance is expected when combining the proposed dynamic model with advanced optimization algorithms to derive the optimal bioprocess operating strategy. [ABSTRACT FROM AUTHOR]

Aims Flocculent wine yeasts were characterized for the expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes, growth kinetics and physicochemical properties of the cell surface during a 6-month sparkling wine fermentation period. Methods and Results The expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes was determined by RT- qPCR. The physicochemical characterization of yeast surface properties was evaluated by the microbial adhesion to solvents method. FLO5 gene was the most expressed one and a linear correlation with the flocculent degree was found. Flocculent strains were more hydrophobic than the commercial wine strain EC1118. Conclusions Gene expressions and the ability to face secondary wine fermentation conditions were strain dependent. The importance of FLO5 gene in developing the high flocculent characteristic of wine yeasts was highlighted. Cell surface properties depended on the time of fermentation. Significance and Impact of the Study Better knowledge about the expression of some genes encoding the flocculent phenotype which could be useful to select suitable starter cultures to improve sparkling wine technology was achieved. A step forward in understanding the complexity and strain-specific nature of flocculation phenotype was done. [ABSTRACT FROM AUTHOR]

The budding yeast Saccharomyces cerevisiae is widely used for brewing and ethanol production. The ethanol sensitivity of yeast cells is still a serious problem during ethanol fermentation, and a variety of genetic approaches (e.g., random mutant screening under selective pressure of ethanol) have been developed to improve ethanol tolerance. In this study, we developed a strategy for improving ethanol tolerance of yeast cells based on metabolomics as a high-resolution quantitative phenotypic analysis. We performed gas chromatography-mass spectrometry analysis to identify and quantify 36 compounds on 14 mutant strains including knockout strains for transcription factor and metabolic enzyme genes. A strong relation between metabolome of these mutants and their ethanol tolerance was observed. Data mining of the metabolomic analysis showed that several compounds (such as trehalose, valine, inositol and proline) contributed highly to ethanol tolerance. Our approach successfully detected well-known ethanol stress related metabolites such as trehalose and proline thus, to further prove our strategy, we focused on valine and inositol as the most promising target metabolites in our study. Our results show that simultaneous deletion of LEU4 and LEU9 (leading to accumulation of valine) or INM1 and INM2 (leading to reduction of inositol) significantly enhanced ethanol tolerance. This study shows the potential of the metabolomic approach to identify target genes for strain improvement of S. cerevisiae with higher ethanol tolerance. [ABSTRACT FROM AUTHOR]

Diphthamide is a conserved modification in archaeal and eukaryal translation elongation factor 2 ( EF2). Its name refers to the target function for diphtheria toxin, the disease-causing agent that, through ADP ribosylation of diphthamide, causes irreversible inactivation of EF2 and cell death. Although this clearly emphasizes a pathobiological role for diphthamide, its physiological function is unclear, and precisely why cells need EF2 to contain diphthamide is hardly understood. Nonetheless, the conservation of diphthamide biosynthesis together with syndromes (i.e. ribosomal frame-shifting, embryonic lethality, neurodegeneration and cancer) typical of mutant cells that cannot make it strongly suggests that diphthamide-modified EF2 occupies an important and translation-related role in cell proliferation and development. Whether this is structural and/or regulatory remains to be seen. However, recent progress in dissecting the diphthamide gene network ( DPH1 - DPH7) from the budding yeast S accharomyces cerevisiae has significantly advanced our understanding of the mechanisms required to initiate and complete diphthamide synthesis on EF2. Here, we review recent developments in the field that not only have provided novel, previously overlooked and unexpected insights into the pathway and the biochemical players required for diphthamide synthesis but also are likely to foster innovative studies into the potential regulation of diphthamide, and importantly, its ill-defined biological role. [ABSTRACT FROM AUTHOR]

Cytochrome P450-dependent monooxygenases (P450s) belonging to the CYP93E subfamily catalyze the C-24 oxidation of the triterpene backbone during the biosynthesis of triterpenoid saponins, which are bioactive plant natural products. In our attempts to produce plant triterpenoids in the yeast Saccharomyces cerevisiae , we observed a poor in vivo catalytic efficiency of the Medicago truncatula CYP93E2. To overcome this biosynthetic bottleneck, we screened publicly available plant genome and transcriptome data for CYP93E subfamily members. Six CYP93E orthologs, exclusively from leguminous plant species, were identified and functionally characterized in S. cerevisiae . Despite the high degree of amino acid conservation, the CYP93E orthologs showed large variations in enzymatic efficiency in yeast. The CYP93E9 from Phaseolus vulgaris showed the highest activity and converted ∼80% of the accumulating in vivo produced substrate β-amyrin to the products olean-12-ene-3β,24-diol and probable 3β-hydroxy olean-12-en-24-oic acid, with a catalytic efficiency that was 61 times higher than that of the M. truncatula CYP93E2. In conclusion, we have expanded the list of functional CYP93E orthologs to a total of nine proteins and show that there are large variations in their catalytic efficiencies when expressed in a heterologous host. Although demonstrated here for the CYP93E family involved in triterpenoid saponin biosynthesis, this phenomenon is undoubtedly extendable to other enzyme families involved in natural product synthesis. Hence, screening for homologous enzymes may become a valuable synthetic biologist’s tool for engineering superior production chassis. [ABSTRACT FROM AUTHOR]

Glycyrrhetinic acid (GA) is one of the major bioactive components of the leguminous plant, Glycyrrhiza spp. (Chinese licorice). Owing to GA's complicated chemical structure, its production by chemical synthesis is challenging and requires other efficient strategies such as microbial synthesis. Earlier investigations employed numerous approaches to improve GA yield by refining the synthetic pathway and improving the metabolic flux. Nevertheless, the metabolic role of transporters in GA biosynthesis in microbial cell factories has not been studied so far. In this study, we investigated the role of yeast ATP binding cassette (ABC) vacuolar transporters in GA production. Molecular docking of GA and its precursors, β-Amyrin and 11-oxo-β-amyrin, was performed with five vacuolar ABC transporters (Bpt1p, Vmr1p, Ybt1p, Ycf1p and Nft1p). Based on docking scores, two top scoring transporters were selected (Bpt1p and Vmr1p) to investigate transporters' functions on GA production via overexpression and knockout experiments in one GA-producing yeast strain (GA166). Results revealed that GA and its precursors exhibited the highest predicted binding affinity towards BPT1 (ΔG = −10.9, −10.6, −10.9 kcal/mol for GA, β-amyrin and 11-oxo-β-amyrin, respectively). Experimental results showed that the overexpression of BPT1 and VMR1 restored the intracellular as well as extracellular GA production level under limited nutritional conditions, whereas knockout of BPT1 resulted in a total loss of GA production. These results suggest that the activity of BPT1 is required for GA production in engineered Saccharomyces cerevisiae. [Display omitted] • Molecular docking of BPT1, VMR1, YBT1, NFT1, YCF1 and MRP1. BPT1 scored -10.9 kcal/mol docking score. • Overexpression of BPT1 and VMR1 under different promoters in GA producing strain. • Genomic integration of BPT1 and VMR1in GA producing strain. • BPT1 can restore GA production to normal level under minimal medium conditions. Knock out of BPT1 completely abolishes GA production. • We conclude that BPT1 is required for GA production, although its role is unclear. [ABSTRACT FROM AUTHOR]

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast. [ABSTRACT FROM AUTHOR]

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non- Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces. [ABSTRACT FROM AUTHOR]

Background Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation. Results In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α- amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg-1 and 393 kUmg-1, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL-1), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield. Conclusion An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production. [ABSTRACT FROM AUTHOR]

□ Proton-induced X-ray emission (PIXE) was used to study submerged fermentation of ethanol from jaggery using Saccharomyces cerevisiae as the organism. The effect of CaCO3as a mineral source for the fermentation process was studied and its activity was monitored. A 3 MeV proton beam obtained from a tandem type pelletron accelerator was used for the multielemental analysis to characterize the calcium uptake in fermentation process. Along with the characterization of Ca, K, Fe, Cr, Co, Ni, Cu, and Zn were also identified in this fermentation process, which may be useful to interpret the various metabolic conditions and studying the impact of these elements. The rate of uptake of calcium by the organism was found to be highest at 14 mg/L supplementation, which was contributed to the final ethanol concentration of 57.8 g/L. A maximum uptake rate of 0.304 mg/L/hr was noticed for CaCO3at 14 mg/L supplementation. The present work demonstrates the suitability of PIXE for the determination of various elements in fermentation processes. [ABSTRACT FROM AUTHOR]

Fermentation of pectin-rich biomass with low concentrations of polysaccharides requires some treatment of the pectin, but does not need complete degradation of the polysaccharide to reach maximum ethanol yields. Cull peaches, whole rotten fruits that are not suitable for sale, contain high concentrations of glucose (27.7 % dw) and fructose (29.3 % dw) and low amounts of cellulose (2.8 % dw), hemicellulose (4.5 % dw) and pectin (5.6 % dw). Amounts of commercial saccharification enzymes, cellulase and cellobiase can be significantly decreased and commercial pectinase mixtures can be replaced completely with a single enzyme, pectate lyase (PelB), while maintaining ethanol yields above 90 % of the theoretical maximum. PelB does not completely degrade pectin; it only releases short chain oligogalacturonides. However, the activity of PelB is sufficient for the fermentation process, and its addition to fermentations without commercial pectinase increases ethanol production by ~12 %. [ABSTRACT FROM AUTHOR]

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose-metabolizing pathways. In this study, a novel two-stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non- Saccharomyces microorganism and coupled to a glucose-utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two-stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae-based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300-310, 2014 [ABSTRACT FROM AUTHOR]

Abstract: Proteins from various biological sources previously identified in wine play important roles in the functioning and survival of their producers and may exhibit oenological properties. Yeasts contribute significantly to the protein pool during and after alcoholic fermentation. While the extracellular proteins of Saccharomyces cerevisiae, the main wine yeast species, have been characterised, those of non-Saccharomyces yeasts remain restricted to a few enzymes. A more comprehensive insight into all proteins released during fermentation could improve our understanding of how yeasts survive and interact in mixed culture fermentations. This study aimed to characterise the exo-proteome of Saccharomyces and selected non-Saccharomyces yeasts in pure and mixed cultures in a wine-like medium. While S. cerevisiae completed the fermentation rapidly, Metschnikowia pulcherrima hardly fermented and Lachancea thermotolerans fermented slowly but steadily. In sequential fermentations, the kinetics resembled those of the non-Saccharomyces yeasts for a period before switching to that of S. cerevisiae. Identification of the proteins present in wine at the end of fermentation using mass fingerprinting revealed the large diversity of proteins secreted and the influence of yeast interactions therein. The fermentation kinetics observed could partially be explained by the extent of the contribution of the different yeast to the protein content. [Copyright &y& Elsevier]

In this article, steady-state optimization of the Saccharomyces cerevisiae fermentation process problem is performed revealing the existence of multiple optimum solutions. The globally optimum solution was determined using the NEOS global optimization solver LINDO. A branch and bound strategy (bnb20.m) and the global search and multistart algorithms in the MATLAB global optimization toolbox were successful in determining locally optimum solutions and these results are validated by plotting the objective function against the decision variables. While in some cases all the strategies were successful in obtaining the globally optimum solutions, an example is presented where the most beneficial product value, which is not a stationary point and lies on the feasible boundary, is obtained by the LINDO global optimization solver (but not the other routines) as the globally optimum solution. The Jones-Kompala model was used to model the steady-state of the fermentation process. While several articles have been published demonstrating the existence of nonlinearities and bifurcations in this model, the challenges posed by this model to optimization has never been investigated so far and this work attempts to do so. Both dilution rate and the oxygen mass transfer coefficient were used as the decision variables individually and together. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:917-923, 2013 [ABSTRACT FROM AUTHOR]

Pure selected cultures of Saccharomyces cerevisiae starters are regularly used in the wine industry. A survey of S. cerevisiae populations during red wine fermentations was performed in order to evaluate the influence of oenological additives on the implantation of the inoculated strain. Pilot scale fermentations (500 L) were conducted with active dry yeast (ADY) and other commercial oenological additives, namely two commercial fermentation activators and two commercial tannins. Six microsatellite markers were used to type S. cerevisiae strains. The methodology proved to be very discriminating as a great diversity of wild strains (48 genotypes) was detected. Statistical analysis confirmed a high detection of the inoculated commercial strain, and for half the samples an effective implantation of ADY (over 80 %) was achieved. At late fermentation time, ADY strain implantation in fermentations conducted with commercial additives was lower than in the control. These results question the efficacy of ADY addition in the presence of other additives, indicating that further studies are needed to improve knowledge on oenological additives' use. [ABSTRACT FROM AUTHOR]

Microbial synthesis of nanocomposite is a green chemistry eco-friendly method. It is a branch of nanoscience for biomedical applications that interconnects nanotechnology and microbial biotechnology. It is only recently that microorganisms have been explored as potential biofactories for synthesis of metal nanoparticles and nanocomposites. By this method, the production efficiency of nanoparticles is very high permitting to relatively easy scale-up the process. Biosynthesis of selenium nanocomposites in aerobic conditions by yeast, Saccharomyces cerevisiae, is reported in the present study. Treating sodium selenite solutions with S. cerevisiae cells and rapid reduction leads to the formation of highly stable selenium nanoparticles in solution. UV spectrum, XRD and SEM analysis of the selenium nanocomposites indicated that they are in the range of size from 30 to 100 nm size. The nanocomposites were further analyzed for antimicrobial activity against a panel of nosocomial infection causing pathogenic bacteria, and they exhibited significant antimicrobial activity. [ABSTRACT FROM AUTHOR]

Optimization of four parameters, i.e., zinc (Zn2+), magnesium (Mg2+), manganese (Mn2+) and yeast extract for bioethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP 01 under very high gravity (VHG, 270 g.L-1 of total sugar) conditions was performed using an L9 (34) orthogonal array design. The fermentation was carried out at 30 °C in 500-mL air-locked Erlenmeyer flasks at the agitation rate of 100 rpm and the initial yeast cell concentration in the juice was approximately 5 × 107 cells.mL-1. The results showed that the order of influence was yeast extract > Mn2+ > Zn2+ > Mg2+ and the optimum nutrient concentrations for the ethanol fermentation were Zn2+, 0.01; Mg2+, 0.05; Mn2+, 0.04; and yeast extract, 9 g.L-1. The verification experiments under the optimum condition clearly indicated that the metals and nitrogen supplementation improved ethanol production efficiency under the VHG fermentation conditions. The ethanol concentration (P), yield (Yp/s) and productivity (Qp) were 120.58 ± 0.26 g.L-1, 0.49 ± 0.01 and 2.51 ± 0.01 g.L-1.h-1, respectively, while in the control treatment (without nutrient supplement) P, Yp/s and Qp were only 93.45 ± 0.45 g.L-1, 0.49 ± 0.00 and 1.30 ± 0.01 g.L-1.h-1, respectively. [ABSTRACT FROM AUTHOR]

Use of ionic liquids has provided a potential effective alternative in the conversion of carbohydrates in lignocellulosic materials into femientable sugars for ethanol produc-tion. To evaluate how the remained ionic liquids in the fermentable sugars affect the sub-sequent ethanol fermentation process, the effects of ionic liquid 1-butyl-3-methylimidazo-lium chloride ([Bmim]Cl) in the medium at different concentrations from 10-3 to 1 g L-1 on the morphological structure, growth and ethanol fermentation of the yeast Saccharo-myces cerevisiae AY92022 were investigated and compared with the control. First, the morphological structures of the yeast at different [Bmim]Cl concentrations were ob-served under an optical microscope. The results show that its single cell morphology re-mained unchanged at all [Bmim]Cl concentrations, but its reproduction rate by budding decreased with the [Bmim]Cl concentration increasing. Then its growth during ethanol fermentation process at different [Bmim]Cl concentrations was examined. The results in-dicated that the ionic liquid [Bmim]Cl inhibited the yeast growth. Its specific growth rate during the log phase and bacterial concentration during the stationary phase all decreased with the increase of [Bmim]Cl concentration. Finally, the ethanol fermentation process at different [Bmim]Cl concentrations was investigated and the results demonstrated that the ionic liquid [Bmim]Cl had a negative effect on ethanol production. When the [Bmim]Cl concentration increased, the final ethanol concentration and its yield from the ferment-able sugars decreased, the finally remaining fermentable sugars increased, and it is inter-esting the ethanol specific formation rate at stationary phase remained unchanged at all [Bmim]Cl concentrations. It was also observed that when the [Bmim]Cl in the medium was 103 g L1, the ethanol fermentation process data was almost no different to that of the control. This suggests that the [BmimjCl in the fermentable sugars should be con-trolled below 10 3 g L-1, thus it would not affect the subsequent ethanol fermentation process. [ABSTRACT FROM AUTHOR]

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective. [ABSTRACT FROM AUTHOR]

The objective of this paper is to investigate the technological usefulness of selected industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus and their intraand interspecific hybrids responsible for excessively acidic musts. The stability of yeast fermentation profiles in apple musts was assessed after 90-170 generations, following previous subculturing under aerobic or anaerobic conditions in media with or without L-malic acid. During this study, 35 apple wines produced by wild strains and their segregates were statistically evaluated according to 12 chemical parameters. Although the wines met the official standards for basic chemical parameters, their total acidity was too low. Both the yeasts and their segregates metabolized from 66.3 to 77.0 % of malic acid present in the must. The industrial wine yeasts and their hybrids exhibited marked polymorphism of fermentation profiles in apple must with elevated L-malic acid content. At the same time, the level of demalication activity made it possible to clearly differentiate segregates from the wild strains, which may suggest that malic acid is probably one of the principal factors in the adaptive evolution of yeasts. Our study proves that among industrial wine yeasts, there are both, strains expressing very high stability (Saccharomyces cerevisiae W-13) and labile ones (S. cerevisiae Syrena). The interspecific hybrids S. eerevisiae x S. bayanus showed low stability of technological features, while the intraspecific hybrid of S. cerevisiae preserved its fermentative capacity. The presented results indicate that fermentative stability assessment under environmental stress can help to select the yeast strains best suited for the fermentation of specific musts [ABSTRACT FROM AUTHOR]

Abstract: Co-inoculation of commercial yeast strains with a bacterial starter culture at the beginning of fermentation of certain varietal grape juices is rapidly becoming a preferred option in the global wine industry, and frequently replaces the previously dominant sequential inoculation strategy where bacterial strains, responsible for malolactic fermentation, are inoculated after alcoholic fermentation has been completed. However, while several studies have highlighted potential advantages of co-inoculation, such studies have mainly focused on broad fermentation properties of the mixed cultures, and no data exist regarding the impact of this strategy on many oenologically relevant attributes of specific wine yeast strains such as aroma production. Here we investigate the impact of co-inoculation on a commercial yeast strain during alcoholic fermentation by comparing the transcriptome of this strain in yeast-only and in co-inoculated fermentations of synthetic must. The data show that a significant number of genes are differentially expressed in this strain in these two conditions. Some of the differentially expressed genes appear to respond to chemical changes in the fermenting must that are linked to bacterial metabolic activities, whereas others might represent a direct response of the yeast to the presence of a competing organism. [Copyright &y& Elsevier]

The ability of baker's yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains. [ABSTRACT FROM AUTHOR]

Alcoholic fermentation (AF) conducted by Saccharomyces cerevisiae has been exploited for millennia in three important human food processes: beer and wine production and bread leavening. Most of the efforts to understand and improve AF have been made separately for each process, with strains that are supposedly well adapted. In this work, we propose a first comparison of yeast AFs in three synthetic media mimicking the dough/wort/grape must found in baking, brewing, and wine making. The fermentative behaviors of nine food-processing strains were evaluated in these media, at the cellular, populational, and biotechnological levels. A large variation in the measured traits was observed, with medium effects usually being greater than the strain effects. The results suggest that human selection targeted the ability to complete fermentation for wine strains and trehalose content for beer strains. Apart from these features, the food origin of the strains did not significantly affect AF, suggesting that an improvement program for a specific food processing industry could exploit the variability of strains used in other industries. Glucose utilization was analyzed, revealing plastic but also genetic variation in fermentation products and indicating that artificial selection could be used to modify the production of glycerol, acetate, etc. The major result was that the overall maximum CO2 production rate (Vmax) was not related to the maximum CO2 production rate per cell. Instead, a highly significant correlation between Vmax and the maximum population size was observed in all three media, indicating that human selection targeted the efficiency of cellular reproduction rather than metabolic efficiency. This result opens the way to new strategies for yeast improvement. [ABSTRACT FROM AUTHOR]

Background: Saccharomyces cerevisiae (Baker's yeast) is found in diverse ecological niches and is characterized by high adaptive potential under challenging environments. In spite of recent advances on the study of yeast genome diversity, little is known about the underlying gene expression plasticity. In order to shed new light onto this biological question, we have compared transcriptome profiles of five environmental isolates, clinical and laboratorial strains at different time points of fermentation in synthetic must medium, during exponential and stationary growth phases. Results: Our data unveiled diversity in both intensity and timing of gene expression. Genes involved in glucose metabolism and in the stress response elicited during fermentation were among the most variable. This gene expression diversity increased at the onset of stationary phase (diauxic shift). Environmental isolates showed lower average transcript abundance of genes involved in the stress response, assimilation of nitrogen and vitamins, and sulphur metabolism, than other strains. Nitrogen metabolism genes showed significant variation in expression among the environmental isolates. Conclusions: Wild type yeast strains respond differentially to the stress imposed by nutrient depletion, ethanol accumulation and cell density increase, during fermentation of glucose in synthetic must medium. Our results support previous data showing that gene expression variability is a source of phenotypic diversity among closely related organisms. [ABSTRACT FROM AUTHOR]

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris. [ABSTRACT FROM AUTHOR]

Abstract: The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from “Primitivo” grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application. [Copyright &y& Elsevier]

The physicochemical properties of tarhana soup produced with different dough treatments, fermentation times, and preservation methods were examined. Tarhana doughs were prepared with yogurt (control) or baker's yeast (Saccharomyces cerevisiae) and fermented for 3 days. Samples were taken at 24, 48, and 72 hr. Samples were then preserved via one of four methods: sun dried, dried in the shade, vacumn dried, and frozen. Frozen samples produced lower organic acid levels after 72 hr of fermentation in both control (0.68 g/100 g) and yeast (0.61 g/100 g) applications than samples that were dried (0.94 g/100 g control samples; 0.81 g/100 g samples with yeast). Increasing fermentation time resulted in a significant effect on the formation of organic acid in the tarhana (p < .01). At 72 hr of fermentation, total acidity increased 11%, 17%, and 23% for tarhana samples vacumn-dried, sun-dried, and dried in the shade, respectively. Preservation methods also affected the moisture, ash, crude protein, total acidity, pH, salt, fat, reducing sugar levels, and the sensory assestment of tarhana soup (p < .01). Sensory characteristics were not significantly affected by baker's yeast in any of the preservation methods used (p > .01). However, sensory scores for tarhana prepared from the samples dried in a sheltered area showed a reduction in color desireablilty as the fermentation time increased. The soup prepared from frozen tarhana (72 hr fermentation, with yeast) had the highest scores with respect to color, mouth feel, flavor, and overall acceptability. Vacuum-dried samples' scores in these areas were also high in comparison to the two other drying methods. [ABSTRACT FROM AUTHOR]