# Trepenoid Metabolic Engineering in Photosynthetic Microorganisms
## 3 Photosynthetic Microorganisms as Terpenoid Production Hosts
### Cyanobacteria  
Cyanobacteria are receiving increased attention as potential photosynthetic production hosts due to their simple cellular organization and their metabolic repertoire, which is characterized by high plasticity [71; 86; 87; 88]. The toolbox of synthetic and systems biology resources for metabolic engineering is rapidly increasing and now includes genome-scale metabolic models [89; 90], transposon libraries [91], CRISPR tools [92; 93; 94], and several genetic synthetic biology parts [71; 95; 96]. Although generation of analysable transgenic lines takes longer than yeast and _E. coli_, they are attractive production hosts due to their photosynthetic growth and relatively simple cellular organization. The first study in cyanobacterial terpenoid engineering involved the production of the hemiterpene isoprene in _Synechocystis_ sp. PCC 6803 [97]. The relatively low initial yield increased fivefold (from 0.05 mg g\({}^{-1}\) to 0.25 mg g\({}^{-1}\)) with the heterologous expression of the MVA pathway [24]. It is interesting to note that the same strategy yielded a much greater, 400-fold increase in isoprene production in _E. coli_[23]. This highlights differential regulation of isoprenoid metabolism between the two types of organism. It is likely that the large increase in _E. coli_ is due to a low innate pathway flux in this organism relative to _Synechocystis_ sp. PCC 6803, which provides a larger dynamic range  
Figure 2: Schematic representation of photosynthetic organisms as heterologous terpenoid biofatories. (**A**) In cyanobacteria and in algal chloroplasts, photosynthesis provides ATP and NADPH, which are, in turn, used to fix carbon dioxide. Glyceraldehyde 3-phosphate (G3P) links carbon fixation with the rest of metabolism. Pyruvate (PYR) is produced from G3P via glycolysis and these two metabolites are the precursors for the MEP pathway. (**B**) In eukaryotic algae, mitochondria produce acetyl-CoA (Ac-CoA) from which the MVA pathway is initiated and IPP/DMAPP are produced. Chloroplasts also contribute to the terpenoid precursor pool. Metabolite exchange between cellular compartments, which is not yet fully elucidated, makes terpenoid metabolism more complex. for improvement. It may also reflect availability of central carbon intermediates to feed into the pathways, differential enzyme behavior in the different organisms, or different availability of DMAPP for isoprene synthase. Isoprene production is limited by the amount of the isoprene synthase expressed. Fusion of the isoprene synthase with the c-phycocyanin beta subunit, which is a highly expressed protein, provided a yield increase to 5.4 mg g\({}^{-1}\)[98]. Overexpression the IPP isomerase to help balance availability of DMAPP conferred a further improvement to 12.3 mg g\({}^{-1}\)[72]. A different approach was used in isoprene production in _Synechococcus elongatus_ PCC 7942, which resulted in the accumulation of 1.26 g L\({}^{-1}\) (approximately 540 mg g\({}^{-1}\)) isoprene after three weeks of growth [73]. This is an impressive outcome in engineered cyanobacteria and by far the highest titres to date. In this work, [PERSON] and co-workers focused on enhancing the flux of the MEP pathway by using targeted metabolite analysis to identify bottlenecks and combining several terpenoid enhancement strategies. A similar approach was implemented in _Synechocystis_ sp. PCC 6803 by studying the effect of overexpressing the MEP pathway enzymes in isoprene production [99]. Many (but not all) of the MEP enzymes (DXS, DXR, ispD, ispE, ispF, ispH) as well as the IDI isomerase had a positive effect. The most dramatic effect though was observed when more efficient isoprene synthases were used, which highlights that the true bottleneck lies in the terpene synthase activity. An early effort to engineer monoterpenoid production in _Synechocystis_ sp. PCC 6803, which expresses the codon-optimized beta-phellandrene synthase from _Lavandula angustifolia_ and reports 50 ug beta-phellandrene L\({}^{-1}\)[100].