# Fermentation Strategies for Production of Pharmaceutical Ternenoids in Engineered Yeast
## 5 Factors Affecting Fermentation Process of Pharmaceutical Terpenoids
### Carbon Source  
The selection of fermentation carbon source depends on the precursor pool designed to be used [60]. Ethanol was often fed to fermentation broth as substrate since it can be readily directed to acetyl-CoA formation [69]. It was preferred instead of glucose as carbon source during the production phase due to increased higher titer and yield in the accumulation of many terpenids in _S. cerevisiae_, such as amorpha-4,11-diene (precursor of artemisinin) [60], artemisinin acid [27], \(\beta\)-amyrin [71], limonee [72], geraniol [73], protopanoxadiol [74], and patchoulol [22] (Table 3). In these studies, diauxic yeast fermentation process was performed, in which glucose is consumed in the first stage of cell growth with the simultaneous production of ethanol that is then used in the second stage of cell growth and biosynthesis of the product of interest [72]. However, high ethanol concentration results in cell stress in _S. cerevisiae_. As a response to this situation, ergosterol synthesis is usually stimulated. This metabolic mechanism had improved the availability of the main precursor 2,3-oxidosqualene, resulting in two times higher total triterpenoid productivities and 2.4-fold increase in carbon yield in ethanol pulse fed fermentations, compared to glucose fed fermentations [75]. However, the use of ethanol as main carbon source is not feasible for industrial terpenoid production, due to its high cost as compared to other carbon sources like glucose. To date, glucose, which is the most widely used carbon source in bioprocesses, has been also selected as feedstock to reach high titer and yield during fermentation of several terpenids, such as bisabolone [76], zerumbone [77], \(\alpha\)-humulene [4], \(\alpha\)-santalene [61], miltiradiene [78], protopanaxadiol and dammarenediol-II [79], and ginseng Rh2 [35] (Table 3). In addition, the promoters chosen to drive expression of heterologous genes also affect the choice of the substrate. For instance, when gal-promoters were used in the construction of engineered yeast, galactose was required to be included in the medium \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compound** & **Titer** & **Strategy** & **References** \\ \hline Artemisinic acid & 25 g/L & \begin{tabular}{c} Overexpression of ADS, CYP71 AV1, CPR1, CYB5, ALDH1 and ADH1 from \(A\). \\ _amnua_, HEM1, and CTT1 under control of \\ galactose-inducible promoters \\ Deletion of gal80\(\alpha\) \\ Downregulation of ERG9 \\ \end{tabular} & [27] \\ \hline Farnesene & 130 g/L & \begin{tabular}{c} Overexpression of ADA, APK, PTA, \\ NADH+HMGr, Farnesene synthase \\ Deletion of adh1 \(\Delta\), ald6\(\Delta\), spp1\(\Delta\), gal2\(\Delta\), bdh1\(\Delta\) \\ Overexpression of enzymes of the MVA pathway to Erg20 \\ Downregulation of ERG9 \\ \end{tabular} & [26] \\ \hline Bisabolone & \textgreater{}900 mg/L & \begin{tabular}{c} Overexpression of tHMG1, ERG20, upc2-1 and bisabolene synthase \\ Downregulation of ERG9 \\ \end{tabular} & [66] \\ \hline Alpha-Santalene & 92 mg/L & \begin{tabular}{c} Overexpression of tHMG1 \\ Deletion of lpp1\(\Delta\), dpp1\(\Delta\) \\ Downregulation of ERG9 \\ \end{tabular} & [61] \\ \hline Patchoulol & 42.1 mg/L & \begin{tabular}{c} Overexpression of ERG20 and PatTps177 \\ Downregulation of ERG9 \\ \end{tabular} & [67] \\ \hline (S)-Linalool & 0.26 mg/L & \begin{tabular}{c} Overexpression of Erg20 and (S)-linalool synthase \\ Diploid \\ \end{tabular} & [68] \\ \hline Geraniol & 1.69 g/L & \
\begin{tabular}{c} 2\(\upmu\) plasmid of PTEF1+VoGES-(GGGS)-ERG20 WW fusion protein \\ 2\(\upmu\) plasmid of PTEF1+HMG1, PPGK1-IDI1, PTEF1- upc2.1 \\ PHCTI-ERG20, oye2\(\Delta\) \\ \end{tabular} & [69] \\ \hline Sabinene & 1.75 mg/L &
\begin{tabular}{c} 2\(\upmu\) plasmid of PTDH3-ERG20 (P96W-N127W)- sabinene synthase (_Salvia_ \\ _pomifera_) fusion protein \\ Diploid ERG9/erg9, ERG20/erg20, \\ PGal1-HMG2 (K6R), PTDH3-HMG2 (K6R) \(\times\) 2 \\ \end{tabular} & [70] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Cont.