Phosphoglycerate mutase 1 regulates dNTP pool and promotes ...
Project Sun to Alcohol2009.igem.org/files/poster/Uppsala-Sweden.pdf · Krebs Cycle Calvin Cycle 3x...
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Institutionen for genetik patologi ENo PK adh Pdc PGM adh2 PDC kivd RuBisCO Enolase Pyruvate Kinase Alcohol Dehydrogenase Pyruvate decarboxylase Phosphoglycerate Mutase Alcohol Dehydrogenase 2 Pyruvate Dehydrogenase Complex Alpha-ketoisovalerate decarboxylase Ribulose-1,5-Bisphosphate Carboxylase Oxygenase PGM ENO Krebs Cycle Calvin Cycle 3x PK PDC CO 2 Ribulose 1,5-bispohsophate + 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate Acetaldehyde Ethanol 3-Phosphoglycerate CO 2 B i o s y n t h esis A m i n o a c i d 2-keto-isovalerate Isobutanal CO 2 Isobutanol kivd RuBisCO Pdc adh Native Pathway Introduced Pathway adh2 Inhibtion: As pyruvate is a precursor of both pathways, we engineered two different approaches to increase the pyruvate levels in order to finally obtain more product by redirecting the carbonflux. The antisense RNA approach targets the formation of the PDC complex . We constructed twelve different antisense RNA which are expressed under copper-inducible promoter pPetE and terminated with T0015. As pirin proteins are known inhibitors of the PDC in other organisms and is known to inhibit growths in Synechocystis sp PCC 6803, our second approach is the protein mediated regulation of the PDC by pirA and pirB.[3][4] Ethanol Module T pPetE RBS B0034 RBS B0034 pdc K273000 adhII (Y) K273001 B0015 K273019 Ethanol Construct: Pyruvate is converted by a pyruvate decarboxylase from Z. mobilis to acetaldehyde. A further reaction performed by the alcohol dehydrogenase 2 from S. cerevisiae leads to ethanol production. Expression of the construct in Synechocystis sp PCC6803 is regulated by the copper inducible promoter pPetE. Butanol Module T pPetE RBS B0034 RBS B0034 kivd K273006 adhII K273005 B0015 K273019 Butanol Construct: We expect a broad range of branched-chain higher alcohols, thus we exemplary demonstrate the mechanism with the pathway for isobutanol production. The enzyme alpha-ketoisovalerate decarboxylase from L. lactis sp. converts keto acids to aldehydes which then are processed to alcohols by adh2 from S. cerevisiae. [2] Results: Even tough the measurements for alcohol concentration were taken with a spectrophometer and thus show a high variance, still a clear difference between induced transformed, non-induced and wild-type E. coli cultures can be observed. Hence we conclude that our constructs are functional. The IPTG inducible promoter for testing purposes in E. coli was pLac (R0011). Results Inhibition Approaches: Nine of the twelve antisense RNA’s were found to have no significant effect on the growth rate while two could not be characterized yet due to very slow growth, even tough transcription has not been induced. Further research will be done. Visit: www.uppsalaigem.org Students: Anders Kristoffersson Erik Florman Jonatan Halvardson Karl Brune Ruiqing Ni Advisors: Thorsten Heidorn Daniel Camsund Mats Wallden Abstract: In the long run our crude oil resources will be on the decline but most importantly the effects of the climate change demand a quick shift to a sustainable fuel economy. Approaching biofuel production by direct synthesis from sunlight has the potential to solve the problems that arise with the conventional fermentation of starches and sugars such as the direct competition of fuel feedstock with food crops.[1] Thus we investigated the production of ethanol and butanol with the use of the cyanobacteria Synechocystis sp PCC6803. Also known as blue-green algae, cyanobacte- ria possess the ability to directly convert sunlight into biofuels. We engineered constructs for ethanol and butanol production as well as strategies to increase the yields of photosynthetic ethanol production. References: [1] Scientific America May 2009, Could food shortage bring down civilization. LR Brown [2] Nature Vol 451|3 January 2008, Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Atsumi et al [3] FEBS Letters 574 (2004) 101-105, A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions. Hihara et al [4] Journal of Bacteriology, January 2007, p. 109-118, Vol. 189, No.1 Pirin regulates pyruvate catabolism by interacting with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity. Po-Chi Lai et al Project Sun to Alcohol Booze Bugs Uppsala iGEM Team 2009
Transcript of Project Sun to Alcohol2009.igem.org/files/poster/Uppsala-Sweden.pdf · Krebs Cycle Calvin Cycle 3x...
Institutionen for
genetik patologi
ENoPKadhPdcPGMadh2PDCkivdRuBisCO
EnolasePyruvate KinaseAlcohol DehydrogenasePyruvate decarboxylasePhosphoglycerate MutaseAlcohol Dehydrogenase 2Pyruvate Dehydrogenase ComplexAlpha-ketoisovalerate decarboxylaseRibulose-1,5-Bisphosphate Carboxylase Oxygenase
PGM
ENO
KrebsCycle
CalvinCycle3x
PK
PDC
CO2
Ribulose 1,5-bispohsophate
+
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetaldehyde
Ethanol
3-Phosphoglycerate
CO2
BiosynthesisAminoacid
2-keto-isovalerate Isobutanal
CO2
Isobutanol
kivd
RuBisCO
Pdcadh
Native PathwayIntroduced Pathway
adh2
Inhibtion: As pyruvate is a precursor of both pathways, we engineered two di�erent approaches to increase the pyruvate levels in order to �nally obtain more product by redirecting the carbon�ux.
The antisense RNA approach targets the formation of the PDC complex . We constructed twelve di�erent antisense RNA which are expressed under copper-inducible promoter pPetE and terminated with T0015.
As pirin proteins are known inhibitors of the PDC in other organisms and is known to inhibit growths in Synechocystis sp PCC 6803, our second approach is the protein mediated regulation of the PDC by pirA and pirB.[3][4]
Ethanol Module
TpPetE
RBS
B0034
RBS
B0034
pdc
K273000
adhII (Y)
K273001 B0015K273019
Ethanol Construct: Pyruvate is converted by a pyruvate decarboxylase from Z. mobilis to acetaldehyde. A further reaction performed by the alcohol dehydrogenase 2 from S. cerevisiae leads to ethanol production. Expression of the construct in Synechocystis sp PCC6803 is regulated by the copper inducible promoter pPetE.
Butanol Module
TpPetE
RBS
B0034
RBS
B0034
kivd
K273006
adhII
K273005 B0015K273019
Butanol Construct: We expect a broad range of branched-chain higher alcohols, thus we exemplary demonstrate the mechanism with the pathway for isobutanol production. The enzyme alpha-ketoisovalerate decarboxylase from L. lactis sp. converts keto acids to aldehydes which then are processed to alcohols by adh2 from S. cerevisiae. [2]
Results: Even tough the measurements for alcohol concentration were taken with a spectrophometer and thus show a high variance, still a clear di�erence between induced transformed, non-induced and wild-type E. coli cultures can be observed. Hence we conclude that our constructs are functional. The IPTG inducible promoter for testing purposes in E. coli was pLac (R0011).
Results Inhibition Approaches: Nine of the twelve antisense RNA’s were found to have no signi�cant e�ect on the growth rate while two could not be characterized yet
due to very slow growth, even tough transcription has not been induced. Further research will be done. Visit: www.uppsalaigem.org
Students:Anders Kristo�erssonErik FlormanJonatan HalvardsonKarl BruneRuiqing Ni
Advisors:Thorsten HeidornDaniel Camsund Mats Wallden
Abstract: In the long run our crude oil resources will be on the decline but most importantly the e�ects of the climate change demand a quick shift to a sustainable fuel economy. Approaching biofuel production by direct synthesis from sunlight has the potential to solve the problems that arise with the conventional fermentation of starches and sugars such as the direct competition of fuel feedstock with food crops.[1] Thus we investigated the production of ethanol and butanol with the use of the cyanobacteria Synechocystis sp PCC6803. Also known as blue-green algae, cyanobacte-ria possess the ability to directly convert sunlight into biofuels. We engineered constructs for ethanol and butanol production as well as strategies to increase the yields of photosynthetic ethanol production.
References: [1] Scienti�c America May 2009, Could food shortage bring down civilization. LR Brown[2] Nature Vol 451|3 January 2008, Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Atsumi et al[3] FEBS Letters 574 (2004) 101-105, A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions. Hihara et al[4] Journal of Bacteriology, January 2007, p. 109-118, Vol. 189, No.1 Pirin regulates pyruvate catabolism by interacting with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity. Po-Chi Lai et al
Project Sun to AlcoholBooze Bugs
Uppsala iGEM Team 2009
![[XLS]images.nature.com · Web viewLsat_1_v5_gn_1_50300.1 GO:0000015 phosphopyruvate hydratase complex GO:0004634 2-Phospho-D-glycerate Phosphoenolpyruvate + H2O, Gallus](https://static.fdocuments.in/doc/165x107/5ae6276c7f8b9a29048d2aba/xls-viewlsat1v5gn1503001-go0000015-phosphopyruvate-hydratase-complex-go0004634.jpg)
