Team:Marburg/Metabolic
M E T A B O L I C E N G N E E R I N G
Metabolic Engeneering
One mayor criteria 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 also tackle one of the most important topic: the climate change.
Planes are considered to be one of the most environmentally damaging transport devices commonly used (Borken-Kleefeld et al., 2010). A quick estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO2 was released from the flights. To encounter this problem, we chose to set our focus on 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.
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, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase 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 heterologous 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 on based previous results, which showed enhancement the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant 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. The IDI, would convert DMAPP to IPP, which is necessary to balance both pools, especially interesting for overproduction of Farnesene, where a ratio of 1:2 in favour of IPP is required.
IspA can synthesise GPP and IPP to 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 potential toxic accumulation. On the other hand, a nontoxic overlay to catch the molecules is required, for example dodecan can be used. (Lee et al., 2017). An alternative supply with CO2 is also required, therefore we copied the system from Lee et al. and introduced a small tube with holes into the Medium.
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). As IDI is an Isomerase, only a comparably medium amount of protein should be sufficient for a enhanced effect, so Promotor BBa_J23110 was chosen. Because the DXS is the a potential bottle neck in Terpene Production, we chose the Promotor BBa_J23111.
Sadly, we weren´t able to test our constructs in vivo and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( BBa_K3228051 and BBa_K3228052).