Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine.

Over recent decades, the average ethanol concentration of wine has increased, largely due to consumer preference for wine styles associated with increased grape maturity; sugar content increases with grape maturity, and this translates into increased alcohol content in wine. However, high ethanol content impacts wine sensory properties, reducing the perceived complexity of flavors and aromas. In addition, for health and economic reasons, the wine sector is actively seeking technologies to facilitate the production of wines with lower ethanol content. Nonconventional yeast species, in particular, non-Saccharomyces yeasts, have shown potential for producing wines with lower alcohol content. These yeast species, which are largely associated with grapes preharvest, are present in the early stages of fermentation but, in general, are not capable of completing alcoholic fermentation. We have evaluated 50 different non-Saccharomyces isolates belonging to 24 different genera for their capacity to produce wine with a lower ethanol concentration when used in sequential inoculation regimes with a Saccharomyces cerevisiae wine strain. A sequential inoculation of Metschnikowia pulcherrima AWRI1149 followed by an S. cerevisiae wine strain was best able to produce wine with an ethanol concentration lower than that achieved with the single-inoculum, wine yeast control. Sequential fermentations utilizing AWRI1149 produced wines with 0.9% (vol/vol) and 1.6% (vol/vol) (corresponding to 7.1 g/liter and 12.6 g/liter, respectively) lower ethanol concentrations in Chardonnay and Shiraz wines, respectively. In Chardonnay wine, the total concentration of esters and higher alcohols was higher for wines generated from sequential inoculations, whereas the total concentration of volatile acids was significantly lower. In sequentially inoculated Shiraz wines, the total concentration of higher alcohols was higher and the total concentration of volatile acids was reduced compared with those in control S. cerevisiae wines, whereas the total concentrations of esters were not significantly different.

Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts.

Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO(2). Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.

Adaptive evolution of Saccharomyces cerevisiae to generate strains with enhanced glycerol production.

The development of new wine yeast strains with improved characteristics is critical in the highly competitive wine market, which faces the demand of ever-changing consumer preferences. Although new strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms strongly limit their use in food and beverage production. We have applied a non-GM approach, adaptive evolution with sulfite at alkaline pH as a selective agent, to create a stable yeast strain with enhanced glycerol production; a desirable characteristic for wine palate. A mutant isolated using this approach produced 41% more glycerol than the parental strain it was derived from, and had enhanced sulfite tolerance. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregated from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains, and highlights the complexity of adaptive responses to sulfite selection.

A novel methodology independent of fermentation rate for assessment of the fructophilic character of wine yeast strains.

The yeast Saccharomyces cerevisiae has a fundamental role in fermenting grape juice to wine. During alcoholic fermentation its catabolic activity converts sugars (which in grape juice are a near equal ratio of glucose and fructose) and other grape compounds into ethanol, carbon dioxide and sensorily important metabolites. However, S. cerevisiae typically utilises glucose and fructose with different efficiency: glucose is preferred and is consumed at a higher rate than fructose. This results in an increasing difference between the concentrations of glucose and fructose during fermentation. In this study 20 commercially available strains were investigated to determine their relative abilities to utilise glucose and fructose. Parameters measured included fermentation duration and the kinetics of utilisation of fructose when supplied as sole carbon source or in an equimolar mix with glucose. The data were then analysed using mathematical calculations in an effort to identify fermentation attributes which were indicative of overall fructose utilisation and fermentation performance. Fermentation durations ranged from 74.6 to over 150 h, with clear differences in the degree to which glucose utilisation was preferential. Given this variability we sought to gain a more holistic indication of strain performance that was independent of fermentation rate and therefore utilized the area under the curve (AUC) of fermentation of individual or combined sugars. In this way it was possible to rank the 20 strains for their ability to consume fructose relative to glucose. Moreover, it was shown that fermentations performed in media containing fructose as sole carbon source did not predict the fructophilicity of strains in wine-like conditions (equimolar mixture of glucose and fructose). This work provides important information for programs which seek to generate strains that are faster or more reliable fermenters.

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy-demanding growth-related processes. Studies using genome-wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.

Production, extraction, and purification of human poly(ADP-ribose) polymerase-1 (PARP-1) with high specific activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in a range of activities associated with DNA metabolism and plays a key role in maintaining the integrity of DNA and chromatin structure. As such, this enzyme is likely to provide a useful target when using a rational drug design approach to develop pharmaceutical reagents, including cancer therapeutics. However, there is still a great deal to learn about the mode of action of PARP-1 and therefore efforts are being directed at gaining a better understanding of the relationship between its structure and function. To this end we have developed a rapid and relatively simple approach to producing and purifying PARP-1. Unlike traditional PARP-1 purification protocols, the method described here requires only one chromatography step thus minimizing losses of the enzyme and also avoids the use of a competitive inhibitor-based affinity chromatography step, which is common to several other protocols in the literature. The product of the method described here is high-quality native PARP-1 with a high specific activity and K(m) and V(max) values similar to what is reported by other workers in the field. This protocol is particularly well suited to making PARP-1 in a quantity and of a quality suitable for structure-function studies.

Analysis of nucleotide methylation in DNA from Corynebacterium glutamicum and related species.

Plasmid DNA (pCSL17) isolated from Corynebacterium glutamicum transformed recipient McrBC+ strains of Escherichia coli with lower efficiency than McrBC- strains, confirming a previous report by Tauch et al. (FEMS Microbiol. Lett. 123 (1994) 343-348) which inferred that C. glutamicum DNA contains methylcytidine. Analysis of nucleotides in C. glutamicum-derived chromosomal and plasmid DNA failed to detect significant levels of methylated adenosine, but methylated cytidine was readily detected. Restriction enzymes which are inhibited by the presence of methylcytidine in their recognition sequence failed to cut pCSL17 from C. glutamicum, whereas enzymes which require methylation at adenosine in GATC sequences failed to cut. Failure of HaeIII to cut two specific sites of C. glutamicum-derived pCSL17 identified the first cytidine in the sequence GGCCGC as one target of methylation in this species, which contains the methyltransferase recognition sequence. Although Brevibacterium lactofermentum-derived DNA showed a similar methylation pattern by HPLC analysis, HaeIII cleaved these GGCCGC sites, suggesting differences in the specificity of methylation between these two species. Results for all analyses of B. flavum DNA were identical to those for C. glutamicum.

Receptors for human interferon alpha on bovine cells: specificity and tissue distribution.

Human interferon alpha 1 (HuIFN alpha 1) is known to protect bovine as well as human cells against viral infection. Hence, we investigated the specificity and tissue distribution of receptors for HuIFN alpha 1 on various cells. [35S]HuIFN alpha 1 bound specifically to homogenates of bovine tissues and particularly to bovine liver, but there was also specific binding to spleen, kidney, brain, adrenal gland, lung, thymus, skeletal muscle, heart, mammary gland and testis. There was no difference in the degree of binding of HuIFN alpha to foetal or adult liver. Competitive binding experiments showed that bovine interferon alpha C (BoIFN alpha C) competed with HuIFN alpha 1 for binding to a bovine liver plasma membrane preparation, indicating that these two IFNs bind to the same receptor. An 35S-labelled IFN alpha 1-receptor complex was isolated from bovine liver extracts by SDS/polyacrylamide gel electrophoresis, and shown to have a molecular weight of 153 kDa. Isolation of the bovine IFN alpha receptor would be a feasible approach to the characterization of the HuIFN alpha receptor.

Structure-function studies of interferons-alpha: amino acid substitutions at the conserved residue tyrosine 123 in human interferon-alpha 1.

Analogs of human interferon-alpha 1 (IFN-alpha 1) were created in vitro by site-directed mutagenesis to investigate the structural requirements at amino acid position 123 for binding to the IFN receptor, antiviral activity, and antiproliferative activity. The tyrosine residue 123, which is conserved in all known mammalian IFNs-alpha and -beta, was replaced by each of 6 amino acids or was deleted from the protein. Several of the substitutions at position 123 partly or completely abrogated antiviral and antiproliferative activities of human IFN-alpha 1 when human or murine cells were used but not when bovine cells were used. However, with analogs in which amino acids structurally related to tyrosine, phenylalanine, or tryptophan were substituted at position 123, there was retention of antiviral and antiproliferative activities using homologous cells. Thus, although there is not an absolute requirement for tyrosine at position 123, conformational changes associated with alterations of this residue are prejudicial to the biological functions of the IFN-alpha molecule.

Amino acid substitutions which alter the antiviral activity of human interferon-alpha 1 on mouse cells.

Human interferon-alpha 1 (IFN-alpha 1) is one of only three human (Hu) IFN-alphas having significant antiviral activity on mouse cells. Specific amino acid substitutions in HuIFN-alpha 1 in the region from amino acids 121 to 136 indicate that this region is critical to the determination of mouse and human cell antiviral activities. Bovine cell activities are relatively unaffected by changes in this region. In particular we have identified the arginine residue at position 125 of human IFN-alpha 1 as a major mediator of the molecules antiviral activity on mouse cells. Various substitutions in the carboxy-terminal region of human IFN-alpha 1 are also evaluated and discussed in the context of recently published data.

An in-vitro study of the role of sucrose and interactions between oral bacteria in possible mechanisms of dental plaque formation.

The adherence of streptococci to hydroxyapatite spheroids (HAS) depended upon the strain used, the fermentable carbon source available during bacterial growth, the number of times a clinical isolate had been subcultured on laboratory media and the pre-treatment of the HAS. Sucrose greatly stimulated the secondary colonization of Streptococcus mutans strain 3209 on HAS pre-equilibrated with this strain, but no similar effect was observed with glucose. Pre-equilibration of HAS with Streptococcus sanguis 7865 inhibited rather than enhanced subsequent colonization by Strep. mutans strain 3209 in the presence of sucrose.