# Trepenoid Metabolic Engineering in Photosynthetic Microorganisms
## 3 Photosynthetic Microorganisms as Terpenoid Production Hosts
### Eukaryotic Algae
#### 3.2.2 Diatoms  
Diatoms are taxonomically distant from higher plants but, being eukaryotic like higher plants, they have both MVA and MEP pathways for isoprenoid precursor production. Diatoms are known to produce high amounts of carotenoids (some of which are high-value and exclusively produced by this group [121]), and a large variety of sterols [121]. Some are also known to emit and contribute a major proportion of marine isoprene [124]. _Thalassioira pseudonona_ and _Phaeodactylum tricornutum_ have emerged in recent years as models for the two main diatom morphological sub-groups (centric and pennate). _Phaeodactylum tricornutum_ is a potentially promising candidate because of its peculiar organization of central carbon metabolism, which comprises three different glycolytic pathways. Besides the Emden Meyerof Parnas glycolysis, _P. tricornutum_ possesses a putative phosphoketolase pathway and a functional eukaryotic Entner-Doudoroff pathway [113]. Although the role of the phosphoketolase pathway and Entner-Doudoroff glycolytic pathways in diatoms is not clear yet, they both produce NADPH and molecules that could possibly enter and fuel the MVA and the MEP pathways. In fact, the putative phosphoketolase pathway possibly produces acetyl-CoA (and 1 ATP and NADPH) and the Entner-Doudoroff pathway yields glyceraldehyde-3-phosphate and pyruvate (and 1 ATP and NADPH). These two pathways, which are naturally present in _P. tricornutum_, have been heterologously expressed and employed by engineered yeasts--where they are absent--for increased availability of isoprenoid precursors [69]. An investigation of the genetic basis of isoprenoid metabolism in _P. tricornutum_ revealed several unusual features including the absence of a conventional squalene epoxidase enzyme and the combination of the enzymatic activities of IPP isomerase and squalene synthase in a fusion enzyme [125]. These unusual characteristics likely indicate that a particular selective pressure has been exerted on these parts of the metabolism. Diatoms benefit from the most advanced genetic toolbox among algae and the availability and the efficiency of the molecular tools to genetically manipulate diatoms has recently progressed enormously. High-efficiency genetic transformation can be achieved by several means ranging from biolistic DNA-coated particle bombardment, electroporation, and bacteria-mediated conjugation in which the latter is compatible with the transfer of large DNA constructs [111]. Methods and resources for transgene expression, RNAi-mediated silencing [126], and CRISPR/Cas9-mediated genome editing [127; 128] have been established. Key to synthetic biology applications, recent advancements have shown that specific centromeric sequences can be added to large episomes to enable extrachromosomal stable maintenance [111], which theoretically allows the simultaneous transformation of very large constructs or multiple genes. This overcomes issues related to the random genomic insertion of the transgenes, which is one of the main bottlenecks in algal genetics transformation. Moreover, recent efforts have succeeded in developing high efficiency targeted genome editing techniques in diatoms [128]. Metabolic engineering efforts in diatoms have been mostly targeted to their lipid metabolism with only a handful of examples of heterologous terpenoid production. However, recent work has highlighted the potential of these organisms. For example, overexpression of the DXS enzyme in the MEP pathway of _P. tricornutum_ resulted in the 2.4 fold increase in the yield (24.4 mg g\({}^{-1}\) DW) of the native high-value carotenoid fucoxanthin [129], which highlights the role of DXS in controlling the flux through the MEP pathway to the carotenoid end-products. DXS is the entry point from central carbon metabolism for the MEP pathway and is the primary rate-limiting step in other organisms used for isoprenoid production. To date, there is only one example of heterologous terpenoid production in diatoms.