Xde

Fig. 4.31 Construction of recombinant DNA plasmid (redrawn from Meaden, 1986).

Fig. 4.31 Construction of recombinant DNA plasmid (redrawn from Meaden, 1986).

expression of the 'foreign' DNA depends on whether or not the DNA becomes integrated into the chromosomes or remains within the plasmid. Although substantially more stable when part of the genome, the copy number (and consequently gene expression) can be low. Conversely if maintained on the plasmid, the expression can be much higher but with poor long-term stability within the cell population.

The advent of recombinant DNA technology matched the then ambitions of brewing geneticists with capability to achieve hitherto impossible goals. Although the vagaries of yeast physiology continued to obstruct some of the more far-fetched concepts, the decade from 1985 proved particularly fruitful in delivering some of the early promise of genetic modification. Table 4.21 links some of the achievements of genetic modification with the 'drivers' and 'targets' established in Table 4.20. The list is by no means exhaustive and, consequently, fails to capture the many excellent reports that detailed similar achievements or were important steps on the way to technical success. Although some modified strains have been subsequently rebadged as 'demonstration GM' projects (Lancashire, 2000), there is no doubting the potential process benefits of, for example, a brewing yeast that circumvents the formation of diacetyl.

With the winds of change and the realisation that commercial exploitation of transformed brewing strains was increasing unlikely, development of new strains has effectively stopped. Indeed, the 1999 EBC contained just one paper on yeast transformation, in this case the construction of a lager yeast which is unable to form sulphite (Francke Johannesen et al., 1999). Today 'genetics' in brewing is about brewing yeast and fermentation Examples of genetic transformation of brewing yeast.

Driver

Target

Transformation of yeast Reference

Vessel utilisation

Efficiency

Vessel utilisation Yeast handling

Vessel utilisation

Beer quality

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

Cost

E.g. P-glucanases, proteases and amylases

Introduction of maltose permease gene increased fermentation rate

Glucoamylase from S. cerevisiae var. diastaticus (also cloned from fungi)

Introduction of yeast flocculation genes (.FLOl)

Bacterial acetolactate decarboxylase (ALDC) which converts a-acetolactate directly to acetoin (bypassing diacetyl formation)

Increasing S02 production by brewing yeast by elimination of the MET10 gene

Capability to manipulate ester synthesis by increasing alcohol acetyltransferase

P-glucanase ex Bacillus subtilis

Secretion of protease

Kodama et al. (1995)

Perry and Meaden (1988)

Watari et al. (1994)

Hansen and KiellandBrandt (1996) Fuji et al. (1994)

Lancashire et al. (1989) Young and Hosford (1987)

differentiation of yeast strains (Section 4.2.6) and identification of bacteria (Section 8.3.4.2). Elsewhere, the technology continues to be exploited, if only in the laboratory. Various publications have described the transformation of non-brewing strains of S. cerevisiae to express a fungal phytase (Han et al., 1999), antifreeze peptides (Driedonks et al., 1995), and to overproduce glycerol (Remize et al., 1999).

4.3.4.3 Legislation, public perception and commercial implementation. In 1986, the Journal of the Institute of Brewing published a series of Centenary Reviews. One by Graham Stewart and Inge Russell titled 'One hundred years of yeast research and development in the brewing industry' captured the excitement of what genetic engineering might offer the brewing industry. In the epilogue to this admirable review, Stewart and Russell (1986) noted that the prediction of future developments was 'a difficult, if not foolish, pastime'. However, as with many applied yeast scientists at the time, the future prognosis for genetic manipulation in the brewing industry was decidedly rosy. Indeed Stewart and Russell (1986) commented that

'the use of manipulated yeast strains in brewing will become commonplace within the next decade with yeast strains specifically bred for such characteristics as extracellular amylases, (3-glucanases, protease, (3-glucosidase production, pentose and lactose utilisation, carbon catabolite derepression (higher productivity) and production of intracellular heterologous proteins (value added spent yeast).'

Given this upbeat introduction it is disappointing that what, at the time, was perfectly reasonable prediction should be so far from the truth. Other than the innate conservatism of the brewing industry, it is not immediately obvious why a genetically modified strain was not used commercially. Certainly, the current media interest and consumer reticence about genetically modified food was not nearly as public or articulate in the early to mid-1990s. Whatever, it is clear that today the odds are stacked against the commercial exploitation of a genetically modified yeast in the brewing industry. Against a background of consumer concern coupled with in a highly competitive market, such an action would be tantamount to commercial suicide!

Within the UK, one genetically modified brewing yeast has been cleared for food use by the appropriate authorities. Hammond (1995) and Walker (1998) have described this daunting and seemingly complex process of approval. The approved yeast was developed by BRi as a 'demonstrator' for the GM technology. The interesting story of the construction and evaluation of this amylolytic brewing yeast can be found in Hammond (1998). Although not a commercial product, BRi periodically produce 'Nutfield Lyte' which continues to be positively received by tasters.

In conclusion, despite the availability of the technology, the numerous hurdles (consumer, media, regulatory) make it almost inconceivable that genetically modified yeast will be used in the brewing industry. The focus is subtly shifting to consideration of exploiting the natural genetic variants that are likely to exist in the yeast population. Although an attractive solution to genetic improvement, the success of such initiatives will depend on the availability of suitably sensitive screening techniques for variants present at very low levels.

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