## 6 Biotechnological Approaches to Increase Steviol Glycos Production
### Metabolic Engineering of Microorganisms  
Most of the SvGls identified so far are extracted from _Stevia reuudiana_. However, the extraction rates of SvGls from the stevia plant are quite low resulting in insufficient supply and high cost. Thus, nowadays, metabolic engineering is being utilized for heterologous production of SvGls in metabolic engineered microorganisms [87]. This had led to tremendous production and commercialization of SvGls in the global market. Metabolic engineering utilizes the methodologies of enzyme engineering, modular synthesis of enzymes, and regulating the gene expression to enhance the production of enzymes/biocatalyst involved in steviol synthesis. Earlier studies reported the construction of yeast cells for de novo synthesis of SvGls, although limited success was attained by using this approach. Recently, researchers have constructed the de novo synthesis of SvGls by modulating the functional expression [79] and overexpressing and altering the 5\({}^{\prime}\) untranslated region (UTR) of key enzymes and ratio of cellular NADPH/NADP+ [88] in the _E. coli_ model system. The findings are indicative that modulating the redox potential may increase the production of SvGls in _E. coli_. _S. cerevisiae_ proved to be another suitable heterologous system for the production of terpenoids by using simple glucose to form steviol for de novo synthesis of SvGls [89]. Specifically, screening and utilizing perfect CYP-CPR homologous pairs seems to be a promising approach for enhancing the SvGls production. In the efforts to increase SvGls production, some researchers have engineered photosynthetic cyanobacteria to produce ent-kaurenoic acid directly from CO2. Engineered _Synechococus elongatus_ strain was used to express the cytochrome P450 enzymes CYP79A1 and CYP71E1, which enabled the synthesis of ent-KA at 2.9 \(\pm\) 0.01 mg L\({}^{-1}\) from CO2 [90]. Glucosyltransferases, in particular, members of the GT1 family, are crucial for glycosylation in the production of the desired rebaudiosides and stevioside. GT1 members use UDP-activated sugars as glycosyl donors, and most of them belong to UDP-dependent glycosyltransferases (UGTs). A whole-cell biocatalysis system was used to express UGT76G1 by constitutive expression system in _S.cerevisiae_. The study deciphers that optimizing the whole-cell response parameters (pH, cell permeability, temperature, glucose concentration) can enhance Reb A. Recently, researchers have developed the pathway for Reb D synthesis by coupling UDP-glucosyltransferase UGTSL2 from _Solanum lycopersicum_ and sucrose syntase StSUS1 from _Solanum tuberosum_ in _E. coli_[91]. The recent advancement to earlier reports is the establishment of a multi-enzyme reaction system to enhance the production of sweeter steviols such as _Reb D_[92]. Recent approaches to increase the production of SvGIs by using different substrates, strains of microorganisms and fermentation methods are discussed in Table 4.  
\begin{table}
\begin{tabular}{p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt}} \hline \hline
**Target Enzyme** & **Strain Used** & **Methodology** & **Production Rate** & **Reference** \\ \hline Ent-kaurene & _E. coli BL21_ (_DE3_) & IPTG (0.02 mM) and fermentation temperature (30 °C), the maximum yield of ent-kaurene was improved & 2.16 mg L\({}^{-1}\) to 194.12 mg L\({}^{-1}\) (shake flask) and 1.872 g L\({}^{-1}\) (in 5-l bioreactor) & [PERSON] et al., 2016 [79] \\ Ent-KA & _E. coli BL21_ (_SSY10_) & Enhanced expression of _KO-Sr_ and optimized fermentation (2.1 mM) concentration & 100.23 mg/L & [PERSON] et al., 2016 [79] \\ Steviol & _E. coli BL21_ (_SSY10_) & Replaced stevia derived KAH by engineering N-terminus of _A. thaliana CYP714A2_ & 15.47 mg/L & [PERSON] et al., 2016 [79] \\  & & UGT module _UGT38C2/_ & & \\ Reb A & _E. coli BL21_ (_SSY10_) & _UGT31D2W/UGT34G1/UGT76G1_ & 10.03 mg/L & [PERSON] et al., 2016 [79] \\  & & incorporated into & 17a_TR29 CYP714A2_ & & \\ Ent-KA & _E.