# Yeast Fermentation for Production of Neutral Distilled Spirits
## 2 Overview of Neutral Distilled Spirits Production
### Yeast Strain Improvement Strategies  
[PERSON] and [PERSON] [PERSON] and [PERSON] (1973) discussed various yeast-strain-engineering strategies for the enhancement of alcoholic fermentation performance in both beverage and biofuel ethanol. For the latter, [PERSON] et al. (2018) discussed approaches to improve yeasts for converting sugar, starch, and cellulosic substrates to bioethanol as a renewable transport fuel. There were similarities in the desired attributes of yeast strains for biofuel and neutral spirit applications. Most notably, this included the minimal biosynthesis of glycerol as well as the other secondary metabolites, such as higher alcohols, acids, and esters. Moreover, the production of a flavour-less distillate in both cases would be a common goal. For both biofuels and distilled spirits, yeast-strain improvement can be accomplished via physiological, genetic, and molecular and synthetic biological strategies. Very high yields of ethanol (>20% _v/v_) have been achieved using cereal starches in large-scale bioethanol (fuel alcohol) distilleries, for example, in the United States. This was possible by employing various yeast physiological and process-engineering approaches, including very-high-gravity fermentation; stress-tolerant yeasts; optimised nitrogen and mineral nutrition; temperature staging; and simultaneous saccharification and fermentation M. (2018). Such approaches have the potential for increasing ethanol yields for neutral spirit producers. The adaptive evolution of yeast strains with improved fermentation characteristics and increased tolerance to stress have also been employed. This involves growing yeast, preferably in chemostat cultures, under a particular selective pressure (e.g., increased ethanol concentrations) to select for faster growing spontaneous mutants. This has proved successful to enhance yeast substrate utilisation, ethanol production, and stress tolerance. Such cell-engineering approaches may be applicable for achieving high ethanol yields for neutral spirit producers. Regarding more directed genetic-based yeast-strain improvements, these involve either classical genetics (e.g., strain hybridisation), genetic engineering (recombinant DNA technology), gene editing (via CRISPR/Cas9), or synthetic biology. Figure 6 summarises some of the strain improvement strategies for industrial yeasts based on molecular genetic approaches that have been discussed by [PERSON] and [PERSON] (2018), for yeasts for bioethanol production. However, the improved yeast traits, as shown in Figure 6, represented the desired characteristics for both biofuel and distilled spirit fermentation. For example, the exploitation of genetically engineered yeast strains to utilise raw starch could circumvent the need to use exogenous amylolytic enzymes to saccharify starch, and such strains were successfully developed (2018). Particularly concerning neutral alcohol, one approach potentially involved a reduction in glycerol biosynthesis, but a suppression of the pathways leading to other by-products were feasible and would be highly beneficial. Regarding glycerol, this represented a significant metabolic product of yeast fermentation that could result in corresponding losses in ethanol yields (around 4%) by _S. cerevisiae_G. (2018). To counteract this, [PERSON] et al. (2018) described how to alter the energy coupling in anaerobic yeast fermentation to reduce or eliminate glycerol biosynthesis whilst, at the same time, maximising alcohol yields. This would also benefit neutral spirit producers. Gene-editing technologies using CRISPR/Cas9 in yeast have been well established [37] and applied to confer ethanol stress tolerance in _S. cerevisiae_[38]. [PERSON] et al. [39] discussed how other industrially relevant traits could be introduced into yeast using gene and pathway engineering.