Beer Brewing Convetions Smart Objectives

Fig. 4.30 Relationship between cell division time and cellular age (redrawn from Barker and Smart. 1996).

The implications of yeast cell ageing in brewery fermentations were studied by Deans el al. (1997). In this elegant work, the yeast cone from a 2000 hi fermenter was fractionated into 5 hi fractions. Each fraction was characterised by age (bud scar analysis and Calcofluor staining, see Section 4.3.3.1) and fermentation performance in tall tube fermenters. As would be anticipated, the age distribution is skewed across the cone, ranging from predominately older cells in early (bottom) fractions to predominately young cells in last cuts at the top of the cone. For example, virgin unbudded cells accounted for 35% of fraction 1 and 72% of fraction 12 whereas cells with 1-2 bud scars represented 42% of fraction 1 and 21% of fraction 14. The significance of these observations is that the fermentation performance of the fractions was found to differ. The mixed age and young yeast fractions fermented more rapidly and attenuated further than the old yeast fractions. Accordingly, the authors suggest a process benefit from more selective cropping procedures that favour mixed-age or young cells. Certainly, it would be anticipated that where feasible there would be benefit for sending more than the initial cone runnings to waste.

The primary driver for cell age research in yeast is to understand the molecular mechanism of the process. In particular, a fundamental question is the interaction between the cell cycle and life span. Jazwinski (1990) has suggested that these events are governed by a circadian clock that integrates metabolism and cell division. A different concept is that of'molecular memory' (Jazwinski, 1990), possibly mediated via 'senescence factors'. These molecules have been postulated to be products of age-specific gene expression, which accumulate in ageing cells resulting eventually in senescence (Egilmez & Jazwinski, 1989). Presumably, to explain the observed variability in cell life span, the 'threshold at which the trigger was set' (Jazwinski, 1990) would vary between yeast cells and strains.

A number of genes have been implicated in yeast longevity. In Louisiana, USA, Jazwinski and co-workers have focused on the /ongevity assurance ^enes (LAG) and RAS genes. LAG1 (D'mello et al., 1994) is found on chromosome VIII and appears to prolong cell age as its transcript declines with the number of generations. However, deletion of LAG1 prolongs the mean life span from 17 to 25 generations. Functionally, these seemingly conflicting results are interpreted as 'this gene directly or indirectly determines or assures the characteristic longevity of the cells' (D'mello et al., 1994). LAG2, found on chromosome XV, has a more direct effect on life span (Childress et al., 1996). Like LAG1, LAG2 is preferentially expressed in young yeast cells. Deletion of LAG2 had no effect on viability, but reduced the mean and maximum life span by 50%. Conversely, over-expression of the gene increased the mean (18 to 20) and maximum (30 to 42) number of generations. Although Childress et al. (1996) could only speculate on the function of LAG2, they suggested an interaction with the RAS-cAMP pathway. As described elsewhere (Section 3.4.2) the RAS genes are important in the cascade that results in the formation of the cellular messenger, cAMP. To emphasise their importance, RAS proteins are present in all eukaryotes from yeast to humans. Work by Sun et al. (1994) proposed that the two RAS genes have opposing effects on the life span of yeast cells. Over-expression of RAS1 had no effect whereas increased expression of RAS2 extended the life span and postponed the senescence-related increase in generation time. Disruption of RAS2 decreased the life span whereas deletion of RAS1 prolonged it! Although an attractive concept, Sun et al. (1994) found that elevated levels of cAMP did not extend but curtailed life span.

Guarente and co-workers at MIT isolated mutants in the wonderfully named UTH ('youth') genes with increased stress resistance and longevity (Austriaco, 1996). More recently the group have used yeast as a vehicle to probe a human disease, Werner's syndrome, that causes premature ageing. This is caused by a recessive mutation to the WRN gene, which has a homologue in yeast, SGS1 (Sinclair et al., 1997). The SGS1

gene is important in yeast cell ageing as its deletion reduced the average life span to about 40% of the wild type strain.

Both the WRN and SGS1 genes code for helicase proteins that are involved in the transcription of the highly reiterated ribosomal DNA in the cell nucleolus. Any mutation that impacts on ribosome formation or structure would potentially effect mRNA translation and protein synthesis. Intriguingly the work of Sinclair et al. (1997) has shown that deletion of the SGS1 gene results in the nucleolus becoming enlarged and fragmented. These events, it is argued, prevent cell division in yeast and, consequently, senescence and death. A subsequent paper (Sinclair & Guarente, 1997) has developed the theme that the nucleolus is the cell's 'Achilles' heel' in ageing. In some respects, this work harks back to the 'senescence factor' theory of Egilmez and Jazwinski (1989). Fragments of DNA (extrachromosomal ribosomal DNA circles or 'ERCs') accumulate as cells become older. These ERCs, which are believed to be byproducts of the cell's ongoing DNA repair processes, may act as a 'clock' for cell ageing.

4.3.3.5 Death and autolysis. Death and autolysis are the final events in the life cycle of yeast. Although cell death (or loss of viability) and, to extent, autolysis can be measured, the triggers for these events are at best empirically understood. In the case of cell viability, programmed cell death through ageing and senescence only account for a fraction of the dead cell population found in fermenter. Clearly, as is well known, the environment of the yeast cell can have a direct impact on cell death. Most notable of the numerous environmental factors is the concentration of the narcotic, ethanol (see Section 3.6.2). The complexity surrounding the assessment of cell death and the mystery of 'vitality' are discussed at length elsewhere (see Sections 7.4.1 and 7.4.2).

It will come as no surprise that yeast is proving a useful vehicle to probe the phenomena of cell suicide in animal systems. Apoptosis (for a review see Matsuyama et al., 1999) or 'programmed cell death' describes the process where in adult humans, some 50-70 billion cells are eradicated on a daily basis! Although the apoptotic pathway found in animal species has not been identified in yeast, S. cerevisiae is being used to gain important insight into these events in higher eukaryotes. Conversely, this vibrant area may throw light on cell death in yeast. For example, a recent report (Madeo et al., 1999) links the accumulation of reactive oxygen species (peroxide, superoxide radicals) with induction of apoptosis in yeast.

Although less glamorous, yeast autolysis has received its fair share of attention. This reflects the impact of cell autolysis on beer flavour and appearance together with its importance as a process in the production of yeast extracts and spreads for human consumption. Autolysis was first observed accidentally by Salkovski in 1989 (Joslyn, 1955). Although considered a process of 'self digestion', the focus of studies on autolysis has been the end result rather than the cellular mechanisms. Suffice to say autolysis is caused by intracellular events mediated by hydrolytic enzymes such as proteinases and glucanases (see Thorn, 1971).

Within brewing, autolysis is triggered by a variety of environmental factors such as temperature, pH, ethanol concentration and osmotic pressure (Lee & Lewis, 1968; Thorn, 1971; Chen et al., 1980; Yamamura et al., 1991). As ever, autolysis is pre vented by good practice in fermentation and yeast handling. The impact of yeast autolysis on beer flavour is dependent on the extent of autolytic damage but, at it worst will give rise to a 'yeast bitten' palate. Autolysis products include low-molecular-weight substances (nucleotides, fatty acids) (Lee & Lewis, 1968; Chen et al., 1980) and high-molecular-weight cell wall material (glucans and mannans) (Lewis & Poerwantaro, 1991). Depending on the concentration, the low-molecular-weight materials affect beer flavour and appearance whereas the cell wall materials can cause haze.

4.3.4 Genetic modification

4.3.4.1 Drivers for strain development. Fundamentally, two drivers have fuelled research into the genetic modification of brewing strains. First, to modify strains to do what they do better, and, second, to modify strains to do what they currently cannot do. Depending on your point of view, the distinction between these two drivers can be narrow. Suffice to say the goal of genetic modification is one of strain improvement enabling yeast to be better equipped or more capable to perform its task of (usually) beer production.

Ironically, brewing yeast strains are poorly equipped to take advantage of what brewing technology in the twenty-first century can offer. Indeed, the physiology, biochemistry and genetics of brewing strains are such that they are now 'rate-limiting' in the introduction of innovative fermentation processes. In other words, we have gone as far as we can with fermentation technology using the current portfolio of brewing strains. To achieve a significant incremental or step change two approaches can be envisaged. First, finding existing strains through natural selection which are better equipped to achieve a desired task, or, second, the introduction of desired genes through genetic manipulation. Although the former approach has undoubtedly been successful in the selection of micro-organisms for the commercial production of enzymes, chemicals, vitamins and pharmaceuticals (Steele & Stowers, 1991), it is the latter approach that this section is focused on. Arguably, this route is quicker and more targeted than the serendipity of natural isolation and selection.

From the mid-1980s, there have been quite stunning developments in the manipulation or modification of yeasts, usually S. cerevisiae. Over this period yeast genetics has galloped ahead through substantial exponential developments in funding, insight and protocols, culminating in the sequencing of the yeast genome (Section 4.3.2.1). Nowadays genetic modification of (primarily) laboratory strains of S. cerevisiae is a routine event such that it is frequently performed by university undergraduates. The focus has moved on to understanding gene function, regulation and integration within cell metabolism.

Inevitably against this background, there have been significant developments in the genetic manipulation of brewing strains. Indeed, a number of generic targets can be identified that have been subject to the attention of brewing yeast geneticists. Articles by Tubb (1981, 1984) at what is now Brewing Research International (BRi), whetted the industry's appetite by painting an appealing picture of what might be possible. Three major targets were identified: (i) enabling yeast to use different carbon sources,

(ii) increasing the efficiency and productivity of fermentation and (iii) improving the control of fermentation and beer quality. With hindsight, the strategy of yeast genetics in brewing has been built around the premise of 'things we currently cannot do' (see Table 4.20). Although relatively easy to identify the 'targets' for genetic change, the 'solutions' are often compromised or incomplete. As with the vastly more significant prizes of understanding cell ageing and human diseases, genetic insight requires to be underpinned by a solid understanding of yeast physiology. This situation was recognised as early as 1991, when Steele and Stowers (1991) noted that 'industry leaders see trained microbial physiologists as being the limiting factor in the development of biotechnology in the coming decade'. Closer to home, Hammond (1995) observed that for brewing yeasts the 'range of improvements possible is still restricted by our lack of knowledge of yeast physiology'.

Table 4.20 General objectives for brewing strain development.

Driver

Objective

Target

Comments

Vessel utilisation

Efficiency

Vessel utilisation

Yeast handling

Vessel utilisation

Beer quality

Cost

Ferment very-high-gravity worts at the normal rate without compromise to beer quality, yeast viability or serial repitching

Ferment non-fermentable wort dextrins

Ferment at elevated temperatures without compromise to beer quality, yeast viability or serial repitching

Modify non-flocculent 'powdery' strain to flocculent strain

Avoidance of diacetyl 'rest' or 'stand' in fermenter or downstream in maturation/ conditioning tank

Modify beer flavour

Avoid the need for addition of exogenous enzymes (processing aids)

Ethanol tolerance Osmotic pressure General yeast physiology

Introduce amylolytic enzymes

Thermotolerance General yeast physiology

Flocculence gene(s)

Introduce foreign enzyme that circumvents the formation of diacetyl or increases flux through pathway

Manipulate specific target genes

E.g. P-glucanases, proteases and amylases

Collection gravity > 1100 Final abv > 10%v/v

Hydolyse linear/branched oligosaccharides ( > G3) to fermentable sugars

Lager fermentations at 20 to 30°C

Move from yeast cropping ex-centrifuge to conventional cone cropping

Depending on the fermentation process, diacetyl stands range from a few days to weeks

Capability to control the concentration of flavour substances, e.g. esters, H2S, SOz

4.3.4.2 Approaches to strain development. It is beyond the scope of this section to consider in any detail the various techniques in use, pre-recombinant DNA technology, by geneticists to modify brewing yeast. Without exception, these approaches are characterised by limitations. The most common and the most problematic was the inability of these methods to selectively effect a single specific change. A good example of the frustrations that this could bring was the introduction of the capability to ferment dextrins into brewing strains. This particular theme has been a popular target of yeast genetics as a 'demonstrator project' for the technology. Early attempts to introduce the glucamylase gene (DEX1 or STA2) from the dextrin utilising wild Saccharomyces (see Section 8.1.3.1), S. cerevisiae var. diastaticus were technically successful in that dextrins were fermented. Unfortunately, S. cerevisiae var. diastaticus has the gene for phenolic off flavour (POF) which, as luck would have it, was adjacent to the gene for glucoamylase. As the early genetic modification methods lacked precision about the bits of DNA being introduced into the recipient strain, the modified yeast produced beers that were phenolic and consequently unacceptable. Although such frustrations could be overcome, the approaches of the early 1980s were rapidly superseded with the advent of recombinant DNA technology. For a user-friendly introduction to the world of the 'early approaches' such as 'mutation and selection', 'hybridisation', 'rare mating', 'cytoduction', and 'spheroplast fusion' the interested reader should consult Brewing Microbiology Hammond (1996).

Recombinant DNA technology brought the vision (Tubb, 1984) of the genetic manipulation of brewing yeast to life. As noted by Meaden (1986) this technique had the major advantage of introducing 'specific genes only', thereby avoiding 'contamination by unwanted or undesirable material from the donor organism'. The details of recombinant DNA technology and application with brewing strains are well beyond the scope of the chapter. General user-friendly overviews are to be found in the 'popular' brewing press (see Lancashire, 1986; 2000; Meaden, 1986; Vakeria, 1991). The interested reader wishing to get into the 'nitty gritty' of genetic modification should consult Walker (1998) or one of John Hammond's excellent reviews on the subject (Hammond, 1996).

The success of recombinant DNA technology with yeast owes much to the relative simplicity and, most importantly, precision with which a desired gene can be inserted into the genome of the target strain. The process is described simplistically below, and by no means does justice to what is an elegant and increasingly sophisticated process. As noted above, the reader is urged to read one of the recommended reviews.

The early part of the process is described diagrammatically in Fig. 4.31 (from Meaden, 1986). The donor DNA is cut into fragments with a restriction enzyme (see Section 4.2.6.1) which is also used to introduce what is usually a single break in a plasmid. These plasmids are small circular DNA molecules, which are often based on the so called '2 (im plasmid' from yeast. The DNA fragments from the donor are mixed with the open plasmid and, the DNA is 'stitched-up' or ligated to form a bank of recombinant plasmids. Transformation of the target strain is achieved by introduction of the plasmid into the yeast cell. The plasmid is then introduced into the cell by removal of the cell wall ('spheroplasting'), treatment with lithium salts or application of an electric current. If transformation is successful, a 'transformant' carrying the desired gene is recovered through a selection process. The stability and the level of t t

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