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− | 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 synthesis add-value compounds with C02 and light as a resource. As a target, we thought about a molecules, which also tackle one of the most important topic: the climate change. | + | 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 CO<sub>2</sub> and light as a resource. As a target, we thought about molecules, which also tackle one of the most important topic: the climate change. |
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− | Plains are considered to be one of the most environmental damaging transport devices commonly used (<a style="padding: 0" href=" https://pubs.acs.org/doi/full/10.1021/es9039693" target="_blank"> Borken-Kleefeld<i> et al.</i>, 2010</a>). A quick estimation suggest, yet alone for the Giant Jamboree in Boston 2018, 14.000.000 CO2 was released for the flights.
| + | Planes are considered to be one of the most environmentally damaging transport devices commonly used (<a style="padding: 0" href=" https://pubs.acs.org/doi/full/10.1021/es9039693" target="_blank">Borken-Kleefeld<i> et al.</i>, 2010</a>). A quick estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO<sub>2</sub> was released from the flights. |
− | To counter this problem, we choose to set our focus on farnesene and limonene, which made up 90 % of the Biojetfuel AMJ700t (50% limonene, 40% farnesene) from Amyris (<a style="padding: 0" href=" https://aem.asm.org/content/aem/81/10/3316.full.pdf" target="_blank">Brennan<i> et al.</i>, 2015</a>). This fuel has proven to be suitable alternative to chemically made Oil based <a style="padding: 0" href=" | + | 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 (<a style="padding: 0" href=" https://aem.asm.org/content/aem/81/10/3316.full.pdf" target="_blank">Brennan<i> et al.</i>, 2015</a>). This fuel has proven to be a suitable alternative to chemically produced, oil based <a style="padding: 0" href=" |
− | https://investors.amyris.com/2012-06-19-Photo-Release-Azul-Brazilian-Airlines-Makes-Successful-Demonstration-Flight-With-Amyris-Renewable-Jet-Fuel-Produced-From-Sugarcane" target="_blank">Jet fuels</a> | + | https://investors.amyris.com/2012-06-19-Photo-Release-Azul-Brazilian-Airlines-Makes-Successful-Demonstration-Flight-With-Amyris-Renewable-Jet-Fuel-Produced-From-Sugarcane" target="_blank">jet fuels</a> |
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− | Farnesene and limonene are both Terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). | + | Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). |
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− | The MEP pathway is a conserved pathway in Bacteria and in photrophic organism, it uses C3-bodys deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodys, Isopentenyl pyrophosphate (IPP) and it´s Iso-mere Dimethylallyl pyrophosphate (DAMPP), are used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous express Limonene synthase to form D-Limonene, where FPP is a substrate for the plant derived Farnesene synthase. | + | 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 (<a style="padding: 0" href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>, 2016</a>). | + | Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in cyanobacteria (<a style="padding: 0" href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>, 2016</a>). |
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− | As a the first step we decided to express heterologous the Limonen and the Farnesen Synthase to establish an overexpression strains. We used the Limonene synthase from <i>Lavandula angustifolia</i> and the Farnesen synthase from <i>Actinidia deliciosa</i> and codon optimised both enymes. | + | 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 <i>Lavandula angustifolia</i> and the Farnesen synthase from <i>Actinidia deliciosa</i> and codon optimised both enzymes. |
− | To redirect the flux into the MEP-Pathway we decided to overexpress the E.coli Proteins DXS, IDI and IspA. These targets were chosen on previous results, that could enhance the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant impairing the grwothrate (<a style="padding: 0" href=" https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0617-8" target="_blank">Choi<i> et al.</i>,2016</a>). | + | To redirect the flux into the MEP-Pathway we decided to overexpress the <i>E.Coli</i> proteins DXS, IDI and IspA. 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 (<a style="padding: 0" href=" https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0617-8" target="_blank">Choi<i> et al.</i>,2016</a>). |
− | This surprisingly suggest, strong production capacities of production in Cyanobacteria.
| + | Surprisingly, this suggest strong capacities of production in Cyanobacteria. |
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− | We chose the DXS, as many pathways are regulated on the first committing step. 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. </p><p> | + | We chose the DXS, as many pathways are regulated on the first committing step. The IDI, would convert DMAPP to IPP, which is necessary to balance both pools, which is especially interesting for overproduction of Farnesene, where a ratio of 1:2 in favour of IPP is required. </p><p> |
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− | IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a Farnesene production strain. | + | 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 (<a style="padding: 0" href=" http://www.jbc.org/content/282/4/2676.long" target="_blank">Xiang<i> et al.</i>, 2016</a>). | + | 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 <i>in vitro</i> (<a style="padding: 0" href=" http://www.jbc.org/content/282/4/2676.long" target="_blank">Xiang<i> et al.</i>, 2016</a>). |
| Also it would be advisable to remove the allosteric feedback regulation of the DXS (<a style="padding: 0" href=" https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161534” target="_blank">Banerjee<i> et al.</i>,2016</a>). | | Also it would be advisable to remove the allosteric feedback regulation of the DXS (<a style="padding: 0" href=" https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161534” target="_blank">Banerjee<i> et al.</i>,2016</a>). |
| </p> | | </p> |
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− | Removal of 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 (<a style="padding: 0" href=" https://pubs.acs.org/doi/10.1021/acs.jafc.7b03625" target="_blank">Lee<i> et al.</i>, 2017</a>). 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. | + | 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. (<a style="padding: 0" href=" https://pubs.acs.org/doi/10.1021/acs.jafc.7b03625" target="_blank">Lee<i> et al.</i>, 2017</a>). An alternative supply with CO2 is also required, therefore we copied the system from Lee <i>et al. </i> and introduced a small tube with holes into the Medium. |
| </p> | | </p> |
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| To balance the pathway we decided to use a weak promotor (<a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23103" target="_blank">BBa_J23103</a>) for the ispA, as competition over FPP could lead to a heavy growth impact (<a style="padding: 0" href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>, 2016</a>). | | To balance the pathway we decided to use a weak promotor (<a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23103" target="_blank">BBa_J23103</a>) for the ispA, as competition over FPP could lead to a heavy growth impact (<a style="padding: 0" href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>, 2016</a>). |
− | As IDI is an Isomerase, only a comparable small amount of Protein should be sufficient for enhanced effect, so Promotor <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a>. Because the DXS is the a potential bottle neck in Terpene Production, we chose the Promotor <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23111" target="_blank">BBa_J23111</a>. | + | As IDI is an Isomerase, only a comparably small amount of protein should be sufficient for a enhanced effect, so Promotor <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a> was chosen. Because the DXS is the a potential bottle neck in Terpene Production, we chose the Promotor <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23111" target="_blank">BBa_J23111</a>. |
| </p> | | </p> |
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− | 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 codon optimised for Synechococcus have been added to the registry ( <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228051" target="_blank"> K3228051</a> and <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228052" target="_blank"> K3228052</a>). | + | Sadly, we weren´t able to test our constructs <i>in vivo</i> 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 ( <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228051" target="_blank"> K3228051</a> and <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228052" target="_blank"> K3228052</a>). |
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