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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 et al. 2010</a>). A quick estimation suggest, yet alone for the Giant Jamboree in Boston 2018, 14.000.000 CO2 was released for the flights. | + | 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. |
− | 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 et al. 2015</a>). This fuel has proven to be suitable alternative to chemically made Oil based <a style="padding: 0" href=" | + | 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=" |
| 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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| 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 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. |
− | 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 et al. 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 … and the Farnesen synthase from … and codon optimised both enymes. | | 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 … and the Farnesen synthase from … and codon optimised both enymes. |
− | 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 et al.2016</a>). | + | 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>). |
| This surprisingly suggest, strong production capacities of production in Cyanobacteria. | | This surprisingly suggest, strong production 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. | | 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. |
| 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 et al. 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 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>). |
− | 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 et al.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 extra cellular 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 et al. 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 End product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extra cellular 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. |
| </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 et al. 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 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>. |
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