# Fermentation Strategies for Production of Pharmaceutical Ternenoids in Engineered Yeast
## 6 Modes of Fermentation Process
### Fed-Batch Mode  
Fed-batch mode is the mostly used operation in the industrial production of diverse type of products as it combines the advantages of both batch and continuous fermentations [103; 118]. It has some advantages over batch mode, such as less inhibitory effect of higher accumulated product and feedstock concentration, higher product formation, extended lifespan of cells, maximum cell viability, and easy control of several important physiochemical factors such as temperature, pH, and oxygen saturation [103; 119]. Other relevant advantages are the possibility of attaining of high cell density, leading to high product formation, and daily product availability. It is generally considered that fed-batch mode with intermittent removal of product and feeding of feedstock when leftover nutrients are exhausted, is the most effective operation mode for terpenids since these products become less toxic within this approach. In addition, this process can achieve the highest volumetric productivity. The most critical parameter in the fed-batch mode is the optimization of feeding since it plays an important role for increasing product yield and productivity [83]. Many pharmaceutical terpenids have been produced by fed-batch fermentation (Table 3). The monoterenes, geraniol [69; 73] and limonene [72], produced by engineered _S. cerevisiae_ strains, have achieved the titer of 1.69 g/L and 0.918 g/L, respectively, with glucose/ethanol mixture or pure ethanol feeding in fed-batch fermentations. These values were quite low (3 to 6-fold lower) when batch fermentations were employed in the same studies. In these works, two strategies were applied to improve productivity of monoterenes during fed-batch fermentation. Firstly, a two-phase fermentation system was employed using the nontoxic extractive solvents dodecane and isopropyl myristate to alleviate the toxicity of geraniol and limonene. Then, the dynamic control of ERG20 was attempted by replacing ERG20 promoter with the glucose sensing HXT1 promoter, which is stronger in the presence of glucose and weaker in the absence of glucose. Thus, replacement of HXT1 promoter provided the diauxic fermentation process, where glucose was consumed during cell growth with ethanol production, which was then used for monoterenes biosynthesis. This approach improved the productivity of geraniol and limonene when glucose/ethanol (1/7), or pure ethanol, were fed instead of glucose as sole carbon source during fed-batch fermentations [69; 72], indicating that the dynamic control of ERG20 by HXT1 promoter could provide flux distribution between cell growth and monoterenes synthesis in the absence or presence of low glucose. Moreover, pure ethanol feeding enhanced monoterene production, since it supplies direct Acetyl-CoA precursors. Apart from monoterenes, great improvement in the production of sesquiterpenes has been achieved using fed-batch processes. For instance, titers of artemisinin precursors amorpha-4,11-diene [60] and artemisinin acid [27] have reached >40 g/L and 25 g/L, respectively, in fed-batch fermentations using highly engineered _S. cerevisiae_. In these studies, the exponential feeding rate for glucose/ethanol mixture substrate feeding and pulse feeding for ethanol substrate (10 g/L) were applied and feeding algorithms were automatically triggered through responses of stir rate, DO percentage as well as evaluation of CO\({}_{2}\) rate [27; 60]. As extracting solvents, isopropyl myristate and methyl oleate were used for artemisinin acid and amorpha-4,11-diene extractions from fermentation broth, respectively [27; 60]. Ref. [60] deleted the GAL1, GAL10, and GAL7 gene cluster, as well as GAL80, to prevent galactose use, which is an expensive inducer, in the production of amorpha-4,11-diene. In this attempt, low level of galactose was required to induce production at the end of batch phase of glucose limited fed-batch fermentation.