# Heterologous Production of Flavour and Aroma Compounds in _Saccharomyces cerevisiae_
## 4 Next Generation Sweeteners
### Steviol Glycosides  
Steviol glycosides (SGs) are a group of active compounds found in the leaves of _Stevia rebaudiana_ Bertoni (also known as \"sweet herb\"), which has been used for centuries as a natural sweetener by native people of South America [89]. The SGs present in stevia plants; stevioside (Ste), rebaudioside(Reb-) A, B, C, D, E, F, and M, duloside A, steviolbioside, and rubusoside, have distinctive sweet tastes that are estimated to range from 30 to 250 times more potent than sucrose [90]. Given these properties, it is not surprising that several SGs are now being used in the food and beverages industries as alternative, low-caloric sweetening agents [91].  
The natural biosynthesis of SGs involves the isoprenoid pathway, with GGPP representing the metabolic branch point from this core metabolic pathway [92]. Four novel enzymatic steps then lead to the production of kaurenoic acid, which is further converted to steviol by kaurenoic acid hydroxylase (KAH). From this point, the activity of a variety of specific UDP-glycostransferases (UGTs) on the basic steviol molecule gives rise to the various SGs that are observed in plant extracts. The above metabolic steps were used to introduce de novo SGs production pathway into _S. cerevisiae_ with five heterologous enzymes driving production of steviol from GGPP (Figure 1).  
Fortunately, the heterologous production of SGs from steviol is aided in yeast by the presence of naturally high levels of cytosolic UDP-glucose [93]. This intracellular UDP-glucose pool can therefore be exploited as a sugar-donor by various heterologously-expressed UDP-glucosyltransferase for the synthesis of specific SGs. Efficient bioconversion (60%) of supplemented stevioside (relative sweetness of \(\sim\)210) to rebaudioside A (relative sweetness of \(\sim\)200), has been recently achieved by expressing the UDP-glycostransferase, UGT76G1, from _S. rebaudiana_ in _S. cerevisiae_[94].  
However, complicating the production of specific SGs is the fact that many glucosyltransferases display loosely-constrained substrate requirements and have the ability to catalyze multiple different glycosylation reactions. This leads to the co-synthesis of mixtures of different SGs, rather than the production of a single pure compound. For example, UGT76G1, which catalyzes the formation of highly desirable steviol glucosides, such as rebaudioside M and D, is also shown to produce undesirable byproducts, including rebaudioside G, Q, I and 1,3-bioside [90]. To address this enzymatic promiscuity, [PERSON] and colleagues [95] used protein site saturation mutagenesis to produce variants in both the predicted substrate-binding pocket, and enzyme active site UGT76G1. A library of 1748 mutants were screened using a strain of _S. cerevisiae_ that carried the set of heterologous genes required for generation of rebaudioside, but lacking a glycosyltransferase [96]. Several mutants, such as UGT76G1 Thr146 Gly and UGT76G1 His155 Leu, displayed the desired increase in rebaudioside M and/or D production with minimal byproduct formation. The study brings important insights into the possibility of altering the enzymatic specificity of steviol glucosyltransferase towards more efficient production of desired SGs.