Katherine A. Smart

The prohibitin family of mitochondrial proteins regulate replicative lifespan

Cellular senescence is determined by multiple factors, including the genetic regulation of metabolism and responses to endogenous and exogenous stresses [1-4]. Recent studies implicate a limited number of gene products in elongating lifespan in yeast and Caenorhabditis elegans [2-4]; these include the C, elegans gene cik-1, a central regulator of metabolism [5], and yeast RAS2, which controls the response to ultraviolet irradiation and other stresses [3]. Another gene postulated to effect senescence is PHB1, the yeast homologue of prohibitin [3], a rodent gene initially identified as a potential regulator of growth arrest and tumour suppressor [6-8]. Highly conserved prohibitin homologues have been identified in mammals [9], Drosophila [10], C. elegans [9], plants [11] and yeast. A second mammalian gene, encoding BAP37, a protein with sequence similarity to prohibitin, is thought to be involved in lymphocyte function [9]. Here, we show that the nuclear-encoded mammalian prohibitin and BAP37 proteins are present in mitochondria, are co-expressed, and interact physically with each other. Deletion of the Saccharomyces cerevisiae homologues, PHB1 and PHB2, results in a decreased replicative lifespan and a defect in mitochondrial membrane potential. Our observations highlight the relationship between the metabolic efficiency of cells and the ageing process, and provide evidence for its evolutionary conservation.

The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation

Individual cells of the yeast Saccharomyces cerevisiae exhibit a finite replicative lifespan, which is widely believed to be a function of the number of divisions undertaken. As a consequence of ageing, yeast cells undergo constant modifications in terms of physiology, morphology and gene expression. Such characteristics play an important role in the performance of yeast during alcoholic beverage production, influencing sugar uptake, alcohol and flavour production and also the flocculation properties of the yeast strain. However, although yeast fermentation performance is strongly influenced by the condition of the yeast culture employed, until recently cell age has not been considered to be important to the process. In order to ascertain the effect of replicative cell age on fermentation performance, age synchronised populations of a lager strain were prepared using sedimentation through sucrose gradients. Each age fraction was analysed for the ability to utilise fermentable sugars and the capacity to flocculate. In addition cell wall properties associated with flocculation were determined for cells within each age fraction. Aged cells were observed to ferment more efficiently and at a higher rate than mixed aged or virgin cell cultures. Additionally, the flocculation potential and cell surface hydrophobicity of cells was observed to increase in conjunction with cell age. The mechanism of ageing and senescence in brewing yeast is a complex process, however here we demonstrate the impact of yeast cell ageing on fermentation performance.

Replicative ageing and senescence in Saccharomyces cerevisiae and the impact on brewing fermentations.

Ageing is the predetermined progressive transition of an individual cell from youth to old age that finally culminates in death. Yeast replicative ageing is a function of the number of divisions undertaken by an individual cell, and may be measured by enumerating the number of bud scars on the cell surface. An aged yeast cell exhibits a distinct morphology and physiology from that of younger cells : the impact of such modifications in cellular function on fermentation performance is examined here. Although the precise mechanism of ageing has not been fully elucidated, the current theories are reviewed and discussed.

Brewing yeast genomes and genome-wide expression and proteome profiling during fermentation.

The genome structure, ancestry and instability of the brewing yeast strains have received considerable attention. The hybrid nature of brewing lager yeast strains provides adaptive potential but yields genome instability which can adversely affect fermentation performance. The requirement to differentiate between production strains and assess master cultures for genomic instability has led to significant adoption of specialized molecular tool kits by the industry. Furthermore, the development of genome‐wide transcriptional and protein expression technologies has generated significant interest from brewers. The opportunity presented to explore, and the concurrent requirement to understand both, the constraints and potential of their strains to generate existing and new products during fermentation is discussed. Copyright

Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation

The fermentable carbohydrate composition of wort and the manner in which it is utilized by yeast during brewery fermentation have a direct influence on fermentation efficiency and quality of the final product. In this study the response of a brewing yeast strain to changes in wort fermentable carbohydrate concentration and composition during full‐scale (3275 hl) brewery fermentation was investigated by measuring transcriptome changes with the aid of oligonucleotide‐based DNA arrays. Up to 74% of the detectable genes showed a significant (p⩽0.01) differential expression pattern during fermentation and the majority of these genes showed transient or prolonged peaks in expression following the exhaustion of the monosaccharides from the wort. Transcriptional activity of many genes was consistent with their known responses to glucose de/repression under laboratory conditions, despite the presence of di‐ and trisaccharide sugars in the wort. In a number of cases the transcriptional response of genes was not consistent with their known responses to glucose, suggesting a degree of complexity during brewery fermentation which cannot be replicated in small‐scale wort fermentations or in laboratory experiments involving defined media. Copyright

Enhanced waterlogging tolerance in barley by manipulation of expression of the N-end rule pathway E3 ligase PROTEOLYSIS6

Summary Increased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N‐end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)‐cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N‐end rule pathway in vitro. Furthermore, we show that Nt‐Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen‐sensor reporter protein MCGGAIL‐GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N‐end rule pathway N‐recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia‐associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia‐related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia‐related genes and phenotypes similar to RNAi lines. We conclude that the N‐end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals.

The oxidative stress response of a lager brewing yeast strain during industrial propagation and fermentation

Commercial brewing yeast strains are exposed to a number of potential stresses including oxidative stress. The aim of this investigation was to measure the physiological and transcriptional changes of yeast cells during full-scale industrial brewing processes with a view to determining the environmental factors influencing the cells oxidative stress response. Cellular antioxidant levels and genome-wide transcriptional changes were monitored throughout an industrial propagation and fermentation. The greatest increase in cellular antioxidants and transcription of antioxidant-encoding genes occurred as the rapidly fermentable sugars glucose and fructose were depleted from the growth medium (wort) and the cell population entered the stationary phase. The data suggest that, contrary to expectation, the oxidative stress response is not influenced by changes in the dissolved oxygen concentration of wort but is initiated as part of a general stress response to growth-limiting conditions, even in the absence of oxygen. A mechanism is proposed to explain the changes in antioxidant response observed in yeast during anaerobic fermentation. The available data suggest that the yeast cell does not experience oxidative stress during industrial brewery handling. This information may be taken into consideration when setting parameters for industrial brewery fermentation.

The impact of catalase expression on the replicative lifespan of Saccharomyces cerevisiae.

The role of catalase on Saccharomyces cerevisiae replicative lifespan was investigated using a wild-type haploid laboratory yeast W303a, a catalase A mutant, a catalase T mutant and an acatalasaemic mutant. Lifespan analysis was performed in two different environmental conditions. Under repressing conditions, on glucose media, catalase T activity, but not catalase A activity was necessary to assure longevity. However, under derepressing conditions, on ethanol media, both catalases were required for longevity assurance. Although catalase activity and carbon source influence yeast lifespan, the relationship between oxidative defence and replicative senescence is complex.

Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol

BackgroundDuring industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs.ResultsThe aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses.ConclusionsOur results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations.

Bio-oil and bio-char from low temperature pyrolysis of spent grains using activated alumina.

The pyrolysis of wheat and barley spent grains resulting from bio-ethanol and beer production respectively was investigated at temperatures between 460 and 540 °C using an activated alumina bed. The results showed that the bio-oil yield and quality depend principally on the applied temperature where pyrolysis at 460 °C leaves a bio-oil with lower nitrogen content in comparison with the original spent grains and low oxygen content. The viscosity profile of the spent grains indicated that activated alumina could promote liquefaction and prevent charring of the structure between 400 and 460 °C. The biochar contains about 10-12% of original carbon and 13-20% of starting nitrogen resulting very attractive as a soil amendment and for carbon sequestration. Overall, value can be added to the spent grains opening a new market in bio-fuel production without the needs of external energy. The bio-oil from spent grains could meet about 9% of the renewable obligation in the UK.