# Fermentation Strategies for Production of Pharmaceutical Ternenoids in Engineered Yeast
## 4 Metabolic Engineering Strategies for Pharmaceutic Terpenoids Production in Yeast  
A brief overview of metabolic engineering applications in _S. cerevisiae_ for terpenoid biosynthesis is herein given. The common metabolic engineering strategies for terpenoids synthesis in yeast, as described in recent reviews [5; 7; 56; 57; 58] can be summarized as such:  
1. Modifying endogenous pathways for synthesis of desired terpenoids. 2. Finding and introducing new heterologous enzymes and pathways into yeast. 3. Determination of rate limiting steps in selected pathways by application of omics studies. 4. Elimination of rate limiting steps in target pathways via overexpression of genes, and cofactor and transporter engineering. 5. Developing enzyme activity and/or specificity by protein engineering. 6. Improving expression level of target key enzymes. 7. Blocking or down regulating competing pathways. 8. Increasing precursor and cofactor supply. 9. Balancing cell growth and terpenoid synthesis for fermentation process. As above mentioned, the direct precursors in sterol pathway, FFP, GPP, GGPP, and squalene epoxide, can be converted into specific terpen through a series of enzymes designated as terpene synthases, which do not exist in _S. cerevisiae_. For this reason, heterologous genes for the functional expression of terpene synthases from plant resources (e.g., taxadiene synthases from _Taxus brevifolia_) or synthetic sources (from NCBI gene accession number) should be introduced into _S. cerevisiae_[57]. On the other hand, Cytochrome P450 (CYPs), which are essential enzymes to modify the hydrocarbon products from terpene synthases, catalyze a wide variety of metabolic reactions, such as oxygenations, deamination, decarboxylation, dealkylation, and C-C cleavage [59]. _S. cerevisiae_ has only three CYPs, which are Erg5 and Erg11 in the ergosterol pathway and Dit2 (putative cytochrome P450 involved in the synthesis of \(N\),_N_-bisformyl dityrosine). For specific or non-native terpenoid production, heterologous expression of CYPs is required. As a successful example, ref. [27] have expressed CYP71 AV1, NADPH dependent cytochrome P450 oxidoreductase (CPR1) and cytochrome b5 (CYB5) from _A. annua_ in _S. cerevisiae_, obtaining 25 g/L of artemisinin acid in optimized fed-batch fermentation. Another strategy is to identify the rate limiting reactions and overexpress the gene, or genes, responsible for these reactions to promote flux into the MVA pathway of _S. cerevisiae_. HMG-CoA reduction to MVA, catalyzed by Hmg1p or Hmg2p, products of HMG1 and HMG2, respectively, was reported as the rate limiting step in the sterol pathway of _S. cerevisiae_. Overexpression of N-terminal truncated HMG1 (tHMG1) and Hmg2p in anaerobic conditions increased squalene titer in this yeast [7; 57]. Moreover, overexpression of the transcriptional factor Upc2-1, constitutively active mutant of Upc2, improved sterols uptake [7]. As a similar strategy, overexpression of all structural genes in MVA pathway (ERG10, ERG13, tHMG1, ERG12, ERG8, IDI1, and ERG20) has enhanced terpenoid production [26; 60]. In addition, down-regulation of downstream genes of competitive pathways can increase the precursor pool. For instance, down regulation of ERG9 in low glucose concentrations through HTX1 promoter, could increase FPP pool in fed-batch fermentations [61]. Acetyl-CoA and NADPH are the essential precursor and cofactor for the biosynthesis of several products (sterols, fatty acids, and polyketides) as well as terpenoids. Overexpression of structural genes in MVA pathway in _S. cerevisiae_ to improve terpenoid synthesis results in draining the acetyl-CoA and NADPH pools, which may negatively affect the cell growth, causing in parallel the decrease in the terpenoid production. Therefore, the increase of cytosolic acetyl-CoA and NADPH pools is required. The most effective way for achieving this was to establish a synthetic pathway. Ref.