# Trepenoid Metabolic Engineering in Photosynthetic Microorganisms
## 3 Photosynthetic Microorganisms as Terpenoid Production Hosts
### Cyanobacteria
Fusion of the beta-phellandrene synthase with the c-phycocyanin beta subunit resulted in beta-phellandrene accumulation at 3.2 mg g\({}^{-1}\)[74]. [PERSON] and co-workers explored the production of the monoterpene limonene and the sesquiterpenes bisabolene in _Synechococcus_ sp. PCC 7002, which achieves yields of 4.0 mg L\({}^{-1}\) and 0.6 mg L\({}^{-1}\), respectively [75]. An increased limonene yield (6.7 mg L\({}^{-1}\)) was achieved in _Synechocystis_ sp. PCC 6803 when two enzymes of the pentose phosphate cycle (the ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase) together with a GPP synthase to increase precursor availability were overexpressed [76]. Sesquiterpenoid production was explored in _Anabaena_ sp. PCC 7120 where farnesene titres reached 0.08 mg g\({}^{-1}\) after two weeks of growth [77]. The highest sesquiterpenoid production reported so far was in _Synechococcus elongatus_ 7942, which reached 19.8 mg L\({}^{-1}\) amorphadiene [78]. The authors achieved this by heterogously overexpressing the _E. coli_ DXS, FPP synthase, and the IPP isomerase. In _Synechocystis_ sp. PCC 6803, squalene, a triterpen derived from FPP by the action of squalene synthase, accumulated to approximately 1.2 mg g\({}^{-1}\) when the downstream squalene hopene cyclase was knocked out [79]. Again, the overexpression of DXS, FPP synthase, and the IPP isomerase resulted in almost 10-fold productivity improvement [78]. The overexpression of the MEP enzymes was implemented in a recent alpha-phellandrine production work in _Synechococcus elongatus_ 7942, which reached 4.6 mg L\({}^{-1}\) alpha-farnesene after seven days of cultivation [80]. Diterpenoid expression in cyanobacteria is less studied. Geranyllinalool, which is a dipterpene alcohol present in tobacco, was produced in _Synechocystis_ sp. PCC 6803 with reported titres of 0.36 mg g\({}^{-1}\)[81]. [PERSON] and co-workers produced 13R-manoyl oxide and reached titres of 0.75 mg L\({}^{-1}\)[101]. Manoyl oxide yield improved to 2 mg L\({}^{-1}\) by codon-optimizing the terpene synthases and expressing them in a self-replicating vector under a strong promoter [82]. As summarized above, production of terpenoids in cyanobacteria has not yielded very high titers so far--high \(\mu\)g L\({}^{-1}\) (or \(\mu\)g g\({}^{-1}\)) to low mg L\({}^{-1}\) (or mg g\({}^{-1}\))--with the notable exception of isoprene production in _Synechococcus elongatus_ (>1 g/L [73]). In an interesting comparison of the isoprene, monoterpene, and sesquiterpenoid yields reported in _E. coli_ and cyanobacteria (normalized to total carbon), [PERSON] et al. note that, in most cases, the cyanobacterial titres are disappointingly low in comparison [102]. The effects of terpenoid engineering on native metabolism are also important to study. In the isoprene production strain developed by [PERSON] et al. [73], the carbon fixation rate of _S. elongatus_ almost doubled. An increased biomass productivity caused by a sink effect has been observed in other metabolic engineering studies (e.g., [103]). Cyanobacteria seem to tolerate the redirection of carbon towards heterologous terpenoid production and increase the flux towards the MEP pathway to compensate for the extra resources consumed [82]. The MEP pathway provides precursors for carotenoids and chlorophyll, which potentially render any significant imbalance due to heterologous terpenoid production unfavorable for growth. Nevertheless, negative effects such as growth retardation and reduction in oxygen evolution have been observed routinely [78; 82; 104]. An interesting observation is that knocking out competing pathways does not seem to improve terpenoid production, which is observed when glycogen synthase and squalene hope cyclase were knocked out in an attempt to enhance limonene and manoyl oxide production, respectively [75; 101]. Many enzymes involved in terpenoid biosynthesis both in Module I (MEP/MVA pathway) and in Module IV (decorations) require electron donors as redox partners.