One mayor criterium to evaluate a chassis is its potential for an application project. To showcase these potential applications, our team decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesize add-value compounds with CO2 and light as a resource. As a target, we thought about molecules which could also help tackling one of the most important issues of our time: climate change.

Planes are considered to be one of the most environment-damaging transport devices (Borken-Kleefeld et al., 2010). A quick estimation suggests, that only for the Giant Jamboree in Boston 2018, 14.000.000 metric tons of CO2 were released due to the teams travelling there by plane. To tackle this problem, we chose to set our focus on the production of farnesene and limonene, which make up 90 % of the biojetfuel AMJ700t (50% limonene, 40% farnesene) from Amyris (Brennan et al., 2015). This fuel has proven to be a suitable alternative to chemically produced, oil based jet fuels.

Metabolic MEP Pathway
Fig. 1- Overview of the MEP-Pathway. Enzymes are marked blue and Green. Limonene and farnesene Synthase are summarised as TPS. Modified after Lin et al 2016

Farnesene and limonene are both terpenoids, derived from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 1). The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, that uses C3-bodies from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodies, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build the longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologously expressed limonene synthase in order to form D-Limonene, whereas FPP is a substrate for the plant derived Farnesene synthase. Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in cyanobacteria (Lin et al., 2016).

As a the first step we decided to heterologously express the limonene and the farnesene synthase to establish overexpression strains. We used the limonene synthase from Lavandula angustifolia and the farnesene synthase from Actinidia deliciosa and codon optimised both enzymes. To redirect the flux into the MEP-Pathway we decided to overexpress the E.Coli proteins DXS (1-deoxy-D-xylulose-5-phosphate synthase), IDI (Isopentenyl-diphosphate Delta-isomerase) and IspA (Farnesyl diphosphate synthase). These targets were chosen based on previous results, which showed increased production of amorpha-4,11-diene to 19.8 mg/L in Synechococcus elongatus PCC 7942 without significantly impairing the growth rate (Choi et al.,2016). Surprisingly, this suggest strong capacities of production in Cyanobacteria and no build-up of toxic intermediates.

Many pathways, such as the MEP-Pathway, are regulated on the first committing step. Therefore we chose the DXS as a first target. IDI isomerase DMAPP and IPP, which is lead to a balance of both pools. For the synthesis of FPP (and ultimately farnesene) a ratio of 1 molecule DMAPP to 2 molecules IPP is required, which disturb the equilibrium. IspA can turn GPP and IPP into FPP, which would improve the efficiency in a farnesene production strain.

To further enhance the efficiency of this pathway, we decided to mutate the DXS-residue 392 from a thyrosine (Y) to a phenylalanine (F). This would lead to a threefold increase in activity in vitro (Xiang et al., 2016). Also it would be advisable to remove the allosteric feedback regulation of the DXS (Banerjee et al.,2016).

Removal of the end product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extracellular diffusion rate is enough to prevent intracellular and potentially toxic accumulation. A nontoxic overlay to catch the molecules would be required, for example dodecan (Lee et al., 2017). Because of this layer, alternative CO2 supplies are required, therefore we made use of the system from Lee et al. and introduced a small tube with holes into the Medium in order to bubble in the CO2.

To balance the pathway we decided to use a weak promotor (BBa_J23103) for the ispA, as competition over FPP could lead to a heavy growth impact (Lin et al., 2016). Because the equilibrium constant for the IDI is around 0.6 in favor of IPP (Diaz et al., 2016), we assumed that only a comparably medium amount of protein should be sufficient for the balancing of both pools and chose Promotor BBa_J23110 to achieve that. The DXS is a potential bottleneck in terpene production, which is why we chose the Promotor BBa_J23111 for it's expression, as this should increase DXS levels.

Sadly, we weren´t able to test our constructs in vivo and had to drop this side project due to time limitations. As of now the submitted parts for the limonene and the Farnesene synthase, which have been codon optimised for S.elongatus UTEX 2973 have been added to the registry (BBa_K3228051 and BBa_K3228052) and we hope that others will continue our efforts to produce more environment friendly bio jetfuels.