# Trepenoid Metabolic Engineering in Photosynthetic Microorganisms
## 3 Photosynthetic Microorganisms as Terpenoid Production Hosts
### Eukaryotic Algae
#### 3.2.1 Chlamydiaomonas reinhardtii  
The green alga _C. reinhardtii_ is historically the longest and most widely used model organism for algal genetics. Efforts in engineering this species have provided proof-of-concept for its suitability as a single-celled factory for the synthesis of many bio-products from recombinant therapeuticenzymes to small molecules. The generation of the mutant _C. reinhardtii_ strains that display to consistently high levels of transgene expression such as UMV4 and UMV11 [122], which greatly facilitated metabolic engineering in this algal species. Its isoprenoid metabolism is based on solely the MEP pathway to provide precursors for photosynthetic pigments and sterols and no complex terpen or terpene-based secondary metabolites have been reported to date. Recent work has shown the potential of using _C. reinhardtii_ to produce the sesquiterpenoid patchoulol by expressing a plant patchoulol synthase gene [83], which is followed by the evaluation of terpene yield in different growth conditions. This reaches the maximum titer of 1.03 mg L\({}^{-1}\) in photoautotrophic growth. By comparing previously published sesquiterpenoid yield in cyanobacteria with those of _C. reinhardtii_, [PERSON] et al. hypothesized that _C. reinhardtii_ might have a relatively larger availability of farnesyl pyrophosphate [75; 83]. However, this work highlighted the availability of preynlphosphate precursors as bottleneck for the heterologous production of terpenoids in _C. reinhardtii_. To address this, terpenoid metabolism was engineered by RNAi-mediated silencing of genes involved in competing pathways that used FPP as a substrate including a squalene synthase and geranyl-geranyl-pyrophosphate synthase. Down-regulation of these competing enzymes resulted in bisabolene yields up to 4.8 mg L\({}^{-1}\). This was further improved to 11.0 mg L\({}^{-1}\) by cultivating the engineered algae in mixotrophic light/dark (16 h/8 h) regimes and providing both 3% CO\({}_{2}\) and acetate in the growth medium [48]. Yields were, however, significantly lower than yields obtained in yeast (more than 900 mg L\({}^{-1}\)) [123]. Very recently, [PERSON] et al. showed that chloroplast localization of diterpenes synthases is essential for the production of diterpenes casbene and manoyl oxide in _C. reinhardtii_. Further carbon flow optimization by fusing the terpene synthases to a mutated GGPP synthase resulted in the production increase to 25 mg L\({}^{-1}\) manoyl oxide [84].  
This work demonstrated the feasibility and efficacy of fine-tuning of isoprenoid metabolism in _C. reinhardtii_ to improve terpenoid yields, but it also underscored that terpenoid production is strongly influenced by cultivation conditions. Therefore, understanding the basis of the endogenous regulation of the isoprenoid pathways will be key toward designing efficient metabolic engineering and bioprocessing strategies. Nuclear transformation in _C. reinhardtii_ occurs by random chromosomal integration of the transgene. Therefore, transgene expression is highly variable across independent cell lines (due to the position-of-integration effects) and may be subjected to currently poorly-understood endogenous silencing mechanisms possibly both at the transcriptional and translational level. Therefore, high-throughput screening platforms are required for identifing the best performing strains. In addition, a targeted gene integration technique would improve reproducibility.