**3. Evolutionary engineering**

#### **3.1 Evolutionary engineering as a simulation of nature**

Evolution is one of the most important processes present in nature to which all living beings are submitted. After traveling around the world collecting much data, Charles Darwin published the book *On the Origin of Species* on the midnineteenth century explaining his theory of evolution based on natural selection. To this day, aside from minor revisions, this theory is the one broadly accepted within the scientific community to best explain evolution. In short, the theory bases itself on the fact that genetic variation occurs among individuals of the same species in a given population leading to phenotypic variations as in morphology, physiology, and behavior traits. In each specific environment, different traits confer different survival rates and different reproduction chances. Upon natural selection, advantageous traits can be passed from generation to generation in a stable heritability. By combining these principles, the progeny of the fittest (best adapted) in a given environment will gradually replace the members of a population and take over. In the case of adverse conditions or sudden environment change, this is one of the main mechanisms on which species rely to keep thriving and avoid extinction.

Evolutionary engineering, also designated as adaptive, directed, or experimental evolution, is an approach where these very same principles of evolution are applied to a selected population in a known and controlled environment [57]. The main difference from nature's evolution is the orientation of the natural selection toward specific selective pressures, the ones which best represent a given environment where we look for an evolution. Over time, individuals initially not optimally adapted may evolve and gain a higher fitness with the accumulation of natural and positive mutations for that specific environment. As the fittest, these individuals will be able to better utilize the available resources, grow faster, and multiply faster in higher number. The natural course is then for their progeny to gradually become dominant within the population, leading to the evolved individuals initially sought. Multiple evolutionary engineering experiments have been performed with different organisms such as *Drosophila* [58], domestic mouse [59, 60], the unicellular

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to be defined.

*3.2.1 Selective pressures*

*3.2.2 Strain choice*

ted to evolutionary engineering.

*Yeast Strain Optimization for Enological Applications DOI: http://dx.doi.org/10.5772/intechopen.86515*

applied to different living beings and contexts.

**3.2 Evolutionary engineering applied to yeast: why and how?**

algae *Chlamydomonas* [61], *Pseudomonas fluorescens* [62], *Escherichia coli* [63], and *S. cerevisiae* [64] demonstrating how the evolution principles can be successfully

Yeast has been the focus of many evolutionary studies due to its potential to generate academic knowledge as well as its broad range of applications. Its success in the evolutionary context is related to different advantages such as the high number of individuals that can be obtained within the same population, the easiness of maintenance/growth of populations with relatively low costs, and a fast generation time. Additionally, evolutionary engineering is a non-GMO technique. As explained before, no direct human manipulation occurs since the yeast itself improves and evolves its genetic background with natural mutations. Thus, evolved yeasts are perfectly safe and can be used in any food and/or beverage context without restrictions. Finally, another positive feature is the simplicity and empirical way on how the evolutionary engineering can be performed [21]. Contrary to other approaches, no genetic characterization or deep knowledge about the selected yeast is required. Nonetheless, when planning a yeast evolutionary engineering, key parameters need

During an evolutionary experiment, the mutations and consequently the diversity generated are completely random and cannot be predicted or controlled [65]. Therefore, it is crucial to identify the selective pressures that will best conduct the selection of positive mutations toward the desired phenotype. Once they are clearly identified, a proper initial experimental characterization should be performed to identify the intensity that these selective pressures should have. If too low there will be no selection, and if too high, yeast will struggle to continue in culture. Additionally, if a further industrial application is predicted, evolutionary engineering should be performed in conditions as close as possible to the actual conditions in which the yeast will later perform. By doing so, yeasts will not only face the selective pressures chosen to drive evolution but also all the other constraints naturally present [66]. For instance, in wine fermentation conditions, yeasts need to cope with stress factors as diverse as low pH, nutrient deprivation, ethanol, osmotic, and oxidative stress which are commonly present [67]. Therefore, it is preferable to use natural or synthetic must as a media that closely mimics realistic conditions while

allowing the control and modification of specific parameters [68].

Depending on the final objective, the choice of a yeast strain can vary. Different laboratory strains have been used in evolutionary approaches with the main goal of generating academic knowledge. On the opposite side of the spectrum, industrial yeast strains have also attracted major interest due to the possibility of improving their efficiency and resistance [9, 66, 69]. Ploidy can also influence strain choice. Haploid strains have the advantage of evolving faster, making it easier to later on identify the mutations that lead to the evolved phenotype [70]. However, they are more sensitive to deleterious mutations that could easily become lethal, whereas diploid strains, as most wine yeast strains, have an increased ability to buffer such mutations. This way diploid strains tend to be more stable and robust when submit*Advances in Grape and Wine Biotechnology*

donor, including the genes of interest.

**3. Evolutionary engineering**

thriving and avoid extinction.

The first step is the selection of a "receptor" strain. This strain possesses a good genetic background and presents numerous properties of interest, except the one aimed to be enhanced. This strain will be crossed with a "donor" strain that possesses the property of interest. A first cross results in a hybrid possessing 50% of the genome of each parental strain. This hybrid is induced to sporulate, and a population of meiotic segregants is constituted. A segregant with the right marker or allele of interest is selected using a simple identification by PCR. This segregant is crossed again with the receptor strain. The second hybrid possesses 75% of the genome of the receptor strain and 25% of the donor strain. Several cycles of breeding/sporulation are performed to regenerate the genome of the receptor strain and to recover its good properties. Generally, four cycles are sufficient and lead to a strain possessing more than 93% of the genome of the receptor strain and less than 7% from the

This approach has been implemented in plants for many years [53, 54]. Its application to the improvement of wine yeasts has started more than 10 years ago [14] and since then it has been applied to generate numerous wine yeast strains The production of H2S, lag phase, and POF character [14], volatile thiol release [55], or

Evolution is one of the most important processes present in nature to which all living beings are submitted. After traveling around the world collecting much data, Charles Darwin published the book *On the Origin of Species* on the midnineteenth century explaining his theory of evolution based on natural selection. To this day, aside from minor revisions, this theory is the one broadly accepted within the scientific community to best explain evolution. In short, the theory bases itself on the fact that genetic variation occurs among individuals of the same species in a given population leading to phenotypic variations as in morphology, physiology, and behavior traits. In each specific environment, different traits confer different survival rates and different reproduction chances. Upon natural selection, advantageous traits can be passed from generation to generation in a stable heritability. By combining these principles, the progeny of the fittest (best adapted) in a given environment will gradually replace the members of a population and take over. In the case of adverse conditions or sudden environment change, this is one of the main mechanisms on which species rely to keep

Evolutionary engineering, also designated as adaptive, directed, or experimental evolution, is an approach where these very same principles of evolution are applied to a selected population in a known and controlled environment [57]. The main difference from nature's evolution is the orientation of the natural selection toward specific selective pressures, the ones which best represent a given environment where we look for an evolution. Over time, individuals initially not optimally adapted may evolve and gain a higher fitness with the accumulation of natural and positive mutations for that specific environment. As the fittest, these individuals will be able to better utilize the available resources, grow faster, and multiply faster in higher number. The natural course is then for their progeny to gradually become dominant within the population, leading to the evolved individuals initially sought. Multiple evolutionary engineering experiments have been performed with different organisms such as *Drosophila* [58], domestic mouse [59, 60], the unicellular

SO2, H2S, and acetaldehyde [56] have been improved using this approach.

**3.1 Evolutionary engineering as a simulation of nature**

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algae *Chlamydomonas* [61], *Pseudomonas fluorescens* [62], *Escherichia coli* [63], and *S. cerevisiae* [64] demonstrating how the evolution principles can be successfully applied to different living beings and contexts.
