# The Terpene Mini-Path, a New Promising Alternative for Ternpenoids Bio-Production
## 3 One Enzyme Access to DMAPP/IPP
### The TMP In Vivo
#### 4.3.2 Yeasts as Host  
Very recently, some reports using the TMP together with an engineered MVA pathway described the production of lycopene, geranylgeraniol, and \(\alpha\)-farnesene in various yeasts (Scheme 10). [PERSON] et al. [33] took advantage of the oleaginous nature of the yeast _Yarrovia lipolytica_ to produce lycopene with the rationale that the lipid body of this yeast species could store more hydrophobic lycopene than the membranes. It was demonstrated that adding the genes coding for choline kinase and IPK results in enhanced intracellular production of IPP/DMAPP upon IOH addition. When the genes coding for lycopene biosynthesis (_crt_E, _crt_B, and _crt_I from _Lamprocystis purpurea_) as well as the gene coding IDI from _PseudoDeslerichu vulgaris_ were introduced in _Y. lipolytica_ strains, enhanced terpenoid precursors content (GPP, PPP, GGPP), as well as higher lycopene titers, were observed for the strain harboring the TMP upon IOH addition. Enhancement of lycopene titer was further observed when lipid synthesis was promoted either by overexpression of _ACC1_ and _DGA1_ (coding, respectively, for an acetyl-CoA carboxylase and a diacylglycerollycerol acyltransferase) or by palmitic acid addition to the culture medium (lycopene titers of \(\sim\)900 and \(\sim\)600 mg/L, respectively, were obtained). In order to improve the utilization of intracellular FPP and GGPP, the introduction of an additional copy of an _idi_ gene and the overexpression of _erg20_, encoding an endogenous FPPS, combined with the addition of palmitic acid in the culture medium, made it possible to reach a lycopene titer of 1.6 g/L in shaken flasks. A final optimization of the palmitic acid concentration (30 mM) and the finding that IOH addition is optimal two days after inoculation led to a final lycopene titer of 4.2 g/L in batch reactor after an 11-day cultivation.  
Using _Saccharomyces cerevisiae_ as a chassis, the production of geranylgeraniol was studied by [PERSON] et al. [34]. The optimization of an initial strain by overexpression of a truncated version of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (_HMBG_1), the use of a GGPPS-FPPS fusion protein, the use of a dodecane layer, and the deletion of the gene _ROX1_ (transcriptional repressor of the sterol pathway) allowed, after 7 days, a final GGGOH titer of 3.66 g/L in a fed-batch cultivation. The introduction of the TMP by overexpression of the _Arabidopsis thaliana ipk_ (the choline kinase being endogenous), as well as the addition of 30 mM IOH after 2 days, improves the final GGGOH concentration to 4.56 g/L (24.6% increase), the latter being further increased to 5.07 g/L upon continuous addition of ethanol (0.35 g/L/h) after day 4.  
[PERSON] et al. used the yeast _Pichia pastoris_ to target the TMP to the peroxisome and produce \(\alpha\)-farnesene [35]. First, the \(\alpha\)-farnesene synthase (\(\alpha\)FS) from _Malus domestica_ was selected from a panel of 20 \(\alpha\)FSs as the enzyme producing the highest concentration in \(\alpha\)-farnesene when introduced in _P. pastoris_ cytoplasm (0.44 g/L). A first optimization of the MVA pathway in the cytoplasm (overexpression of _tHMG1_, _idi1_ and _erg20_) led to a 0.67 g/L titer in \(\alpha\)-farnesene. A further increase to 1.01 g/L was noted when _EcPDH_, the gene coding the pyruvate dehydrogenase from _E. coli_, was overexpressed to increase the acetyl-CoA supply. In comparison, when the gene coding the \(\alpha\)FS from _Malus domestica_ was targeted to the peroxisome, a 0.53 g/L titer in \(\alpha\)-farnesene was observed, increased to 1.32 g/L when the TMP (with IOH addition) was also targeted to the peroxisome as well as _idi1_ and _erg20_. With an extra copy of these two genes, a further increase to 1.69 g/L was observed. Combining both optimizations (cytoplasm + peroxisome) gave a 2.18 g/L titer, and the highest titer of 2.56 g/L in 72 h was attained in shake flasks with carbon source cofeeding (oleic acid, sorbitol).