# Biosynthesis of Edible Terpenoids: Hosts and Applications
## 2 Microbial Chassis as Cell Factories for Edible Terpenoids
### Prokaryotic Microorganisms
#### 2.1.1 E. coli  
_E. coli_, possessing a well-defined genetic background and comprehensive genetic manipulation techniques, serves as a model prokaryotic microorganism. Presently, diverse strategies have been employed to enhance the titer of terpenoids in _E. coli_[41].  
In 2009, [PERSON] et al. optimized and combined the RBS of 20 endogenous genes in _E. coli_ to enhance the flux of the DXP pathway and achieved a 5-fold increase in lycopene production [24]. The multivariate-modular approach to metabolic-pathway engineering strategy was used to optimize the endogenous MEP pathway for IPP synthesis and the heterologous pathway for taxadiene synthesis, achieving a 15,000-fold increase in taxadiene production [23]. Nevertheless, dependence solely on the endogenous MEP pathway remains suboptimal. A study reported that by reconstituting five genes in the MVA pathway and the _idi_ gene in _E. coli_, a three-fold increase in lycopene titer was realized [42]. In recent years, several naturally occurring plant-derived terpenoids have also been biosynthesized in _E. coli_.  
In [PERSON]'s lab, they screened for an active geranyl diphosphate synthetase (GGPS) and fused a truncated leader peptide to promote the soluble expression of geraniol synthetase (GES). The geraniol titer of engineered _E. coli_ increased to 2.12 g/L [25]. In order to alleviate the toxicity of the end product, they introduced an alcohol acyltransferase to esterify geraniol to geranyl acetate, and then they used another _E. coli_ expressing acetyl esterase, which hydrolyzes geranyl acetate to geraniol. By fed-batch fermentation in a 10 L bioreactor, the titer of geraniol was reached to 13.19 g/L [26]. Further, they recruited a truncated linalool dehydatase isomerase (LDI) to isomerize geraniol to linalool and achieve de novo biosynthesis of myrcene at 1.25 g/L by using aerobic-anaerobic two-stage fermentation [27]. In order to efficiently produce plant-derived sesquiterpenes \(\beta\)-elemene in _E. coli_, [PERSON] and his colleagues systematically designed the metabolic pathway. They screened for a germacrene A synthase (NS), which could transform farnesyl pyrophosphate (FPP) to \(\beta\)-elemene via germacrene A, engineered the RBS of NS, and linked NS with farnesyl diphosphate synthase (IspA). After rewriting the native central carbon metabolism of acetyl-CoA and pyruvate and overexpressing two membrane transporters, ToIC and MsbA, to pump compounds extracellularly, they achieved the highest reported titer of 3.52 g/L of \(\beta\)-elemene and 2.13 g/L of germacrene A [28].  
_E. coli_ cell factories can not only elongate carbon chains but also cleave carbon chains. As we know, carotenoid cleavage dioxygenases (CCDs) could catalyze the cleavage and oxidation of \(\alpha\)-carotene or \(\varepsilon\)-carotene to form ionone. [PERSON] et al. used an evolved thiooredoxin-CCD1 fusion protein in a lycopene-producing strain to synthesize \(\alpha\)-ionone. However, they found that the by-product H\({}_{2}\)O\({}_{2}\) of CCD1 will break down the precursor lycopene. So, they overexpressed the alkyl hydroperoxide reductase (_alp_C/F) to reduce oxidative stress. As a result, they achieved 700 mg/L \(\alpha\)-ionone in a 5 L bioreactor [29]. For dihydro-\(\beta\)-ionone, [PERSON] and his colleagues identified an enoate reductase (DBR1) gene from _Artemisia annua_, which can transform \(\beta\)-ionone to dihydro-\(\beta\)-ionone. Then, they constructed a cell-free system containing CCD1, DBR1, and glucose dehydrogenase (GDH) for dihydro-\(\beta\)-ionone production. The molar conversion was 85.8% [30]. Furthermore, they constructed a de novo \(\beta\)-ionone biosynthesis platform in _E. coli_. When co-cultured with _S. cerevisiae_ harboring DBR1, the final yield of dihydro-\(\beta\)-ionone was achieved at 27 mg/L [31].