Introduction

The bread making process is one of the oldest applications of biotechnology. The term bread defines a great variety of baking products, which vary in formulation, ingredients, and processing conditions. For most of them, however, the use of baker's yeast is fundamental to obtaining a high quality product according to the demands of consumers. Baker's yeast contributes substantially to the flavor and crumb structure of bread, but its primary role is the production of CO2, via the alcoholic fermentation of sugars, which results in dough leavening.

Commercial baker's yeasts are domesticated strains of S. cerevisiae that have been selected and optimized for baking applications. Most of them are homothallic, with a high and irregular degree of ploidy and low sporulation ability (1). In addition, they exhibit chromosomal-length polymorphisms and rearranged chromosomes with multiple translocations (2). These special characteristics are the result of natural selection by adaptative evolution together with the successive application of improvement programs. The polymorphism of baker's yeast reflects, at least in part, selection by continuous long term nutrient limitation. In industrial practice, the yeast is grown aerobically under conditions (fed-batch) in which the supply of the carbohydrate feedstock is limited, to avoid ethanol formation. When a yeast population is cultured under this physiological situation, it undergoes a series of adaptative shifts whereby clones that demonstrate better efficiency of nutrient utilization successively replace one another over time (3). The mechanism underlying this response includes random and nonrandom mutations (4) and unequal crossing over, leading to gene amplification, changes in protein structure, and altered expression patterns (5). When compared with laboratory strains of S. cerevisiae, baker's yeasts has more copies of the SUC gene, which appears to be amplified and translocated to several chromosomes (6,7). This gene encodes for invertase, the enzyme hydrolyzing sucrose, the main carbon source found in beet and cane molasses used for industrial feeding. Thus, amplification of SUC genes endows yeast cells with a higher sucrose hydrolyzing ability, overcoming a limiting step for growth and biomass yield (8). A clear example of this evolutive mechanism is the appearance of multiple tandem duplications of high affinity hexose transporters (HXT gene family), in a yeast population grown in a continuous glucose-limited environment (9).

The second factor accounting for many of the characteristics of baker's yeast is the repeated use of improvement strategies that provide a high genetic variability. Strain improvement of baker's yeast has traditionally relied on classical genetic techniques, including random mutagenesis, hybridization, and protoplast fusion. The continuous mating and later segregation of meiotic products in baker's yeasts increase the possibilities of chromosomal reorganizations, both in size and number, leading to polymorphism. This phenomenon is enhanced by the presence of many Ty transposable elements and subtelomeric Y' regions in the baker's yeast genome (7), a key factor for chromosomal rearrangements (10). This feature could have also favored the selection of traits appreciated in baker's yeast, because the MAL loci and the SUC genes are located at the telomeres of this organism (11). The selection for broad traits, as robustness, growing rate, or fermentative capacity would also have contributed to the genetic constitution of baker's yeast. Although there is a lack of knowledge of the genes responsible for these properties, they are favored by a high ploidy level, as evidenced by much experimental data. For example, industrial yeasts show higher levels of glycolytic enzymes than wild-type strains (12). Dough-leavening ability also correlates with the activity of the MAL genes (13,14), which determine the capacity to transport and hydrolyze maltose, the main sugar than sustains dough fermentation. This is certainly not surprising, because duplication of blocks of genes has been a recurring feature of yeast genome evolution (15,16). Increasing the number of chromosomes will increase the number of favorable genes leading to strains with enhanced fitness, increased maltose utilization capacity, and more vigor (17, 18). Thus, natural adaptation and the selective pressure exerted for decades by producers have led to the transformation, at low cost, of an abundant feedstock to cell biomass of high technological quality.

Nevertheless, there are still important traits in baker's yeast that are far from optimal. Tolerance to various stresses, such as osmosis, freezing, or desiccation, is clearly inadequate. Our limited understanding of the physiological and genetic determinants of these important commercial properties suggests that they are under the control of multiple genes and complex regulatory mechanisms (19, 20). Some of these desired phenotypes could also go against biological design (21). Thus, it is unlikely that classical breeding programs could provide further improvement of these characteristics. Given this scenario, the ability to transform baker's yeast by recombinant DNA technology has opened the possibility to manipulate just a single gene or pathway, without altering other key or valuable genes. Its is also possible to knock out undesirable genes, to engineer the constitutive expression of groups of genes involved in a particular function, or even to switch on or off the transcription of a given gene in a specific technological step. The transfer of heterologous genes to S. cerevisiae means that there is now the possibility of using yeast as a cell factory. This has allowed, in the past decade, the development of industrial strains with unsuspected properties, giving a new generation of baker's yeast.

This chapter will collect the available information on the most important advances in the engineering of baker's yeast, from both fundamental and technological aspects. It will also review critically the molecular tools applied for transformation of industrial strains. Information concerning wild S. cerevisiae and other industrial yeasts, like brewing or wine strains, will also be considered where it provides knowledge to address unresolved problems or to reveal future perspectives. To date, most of the recombinant approaches have been used for the modification of traits linked to single or a limited number of genes. Thus, the main targets for strain improvement by genetic engineering can be grouped into three categories: (1) extension of substrate range, (2) heterologous enzyme production, and (3) overproduction of essential nutrients.

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