# Fermentation Strategies for Production of Pharmaceutical Ternenoids in Engineered Yeast
## 6 Modes of Fermentation Process
### Fed-Batch Mode
In addition, same authors applied phosphate limited fed-batch fermentation, which could limit growth of cells and channel carbon flux to product formation, ending up with more than 2 times of amorpha-4,11-diene production. Finally, ethanol-restricted fed-batch fermentation was improved, resulting in 37 g/L of amorpha-4,11-diene, which was lower than ethanol unrestricted fed-batch fermentation but this process required lower levels of oxygen uptake rate (OUR), what is desired and feasible for industrial scale fermentation. On the other hand, ref. [27] used isopropyl myristate solvent to extract artemisinin acid from fermentation and improved its productivity in fed-batch fermentations. With this method, a significant enhancement on titer and yield was observed by eliminating successfully the acid from the aqueous phase, since artemisinin acid precipitated in fermentation broth, complicating sampling and negatively affecting viability of cells. Other pharmaceutical sesquiterpenes, such as bisabolene [76], nerolidol [84], patchoulol [22], zerumbone [77], and \(\alpha\)-humulene [4] were accumulated by engineered \(S\). _cerevisiae_ strains with titers of 5.2 g/L, 5.5 g/L, 0.467 g/L, 0.040 g/L and 1.7 g/L, respectively, in fed-batch fermentations. Constant feeding rate triggered by pH rises was used in bisabolene production of glucose fed-batch fermentation [76] while pulse feeding, which was programmed using script controller feeding and triggered by DO spikes, was performed in nerolidol production [84]. Moreover, carbon-source controlled three-stage fed-batch fermentation was employed for patchoulol production [22], whereas in zerumbone and \(\alpha\)-humulene production, constant feed rate triggered by DO and pH rises was applied [4; 77]. In these studies, combination of constitutive copper-inducible and diauxic-induced transcription regulation patterns was employed to improve nerolidol production in the fed-batch fermentation through enhancing the growth rates in each phase (exponential, ethanol growth, and diauxic phases) and productivities after the diauxic shift. Thus, combined system yielded better nerolidol titer in the carbon-overflow fed-batches than substrate restricted-fed batches. The improvement in nerolidol productivity might be due to MVA pathway response to \"overflow\" metabolism, as is seen in ethanol/acetate/glycerol production through glycolysis [84]. Moreover, a carbon source controlled three stages fermentation (first stage: high glucose concentration feeding; second stage: lower concentration glucose and glycerol feeding; third stage: ethanol feeding) was used to balance the trade-off between the competitive squalene and patchoul pathways in the production of patchoul by engineered \(S\). _cerevisiae_[22]. The pharmaceutical diterpenes, geranylgeraniol [89], miltiradiene [78], and oxygenated taxanes (precursors of paclitaxel) [90] were also accumulated (3.31 g/L, 0.488 g/L and 0.033 g/L, respectively) by genetically modified \(S\). _cerevisiae_ strains (co-culture with \(E\). _coli_ in oxygenated taxanes accumulation) in fed-batch fermentations. In these studies, after depletion or decrease of substrate in the medium, feed rates of 5.6 g/h of glucose/ethanol mixture for geranylgeraniol, 5 mL/h of glucose and other ingredients for miltiradiene, and feed pulses of 20 g/L glucose and 50 g/L xylose for oxygenated taxanes productions were applied. In addition, several triterpenes and their precursors, such as protopanaxacid (1.189 g/L) and dammarenediol-II (1.548 g/L) [79], \(\beta\)-amyrin (0.139 g/L and 0.108 g/L) ([71; 85], respectively), betulinic acid (0.182 g/L) [75] and ginseng Rh2 (2.25 g/L) [35] were attempted to accumulate and successfully produced in fed-batch fermentations. In these studies, distinct feeding strategies were applied.