Project Sun to Alcohol Booze Bugs
Abstract: In the long run our crude oil resources will be on the decline but most importantly the e ects of the climate Uppsala iGEM Team 2009 change demand a quick shift to a sustainable fuel economy. Approaching biofuel production by direct synthesis from Students: Advisors: sunlight has the potential to solve the problems that arise with the conventional fermentation of starches and sugars Anders Kristo ersson Thorsten Heidorn such as the direct competition of fuel feedstock with food crops.[1] Thus we investigated the production of ethanol Erik Florman Daniel Camsund and butanol with the use of the cyanobacteria Synechocystis sp PCC6803. Also known as blue-green algae, cyanobacte- Jonatan Halvardson Mats Wallden ria possess the ability to directly convert sunlight into biofuels. We engineered constructs for ethanol and butanol Karl Brune production as well as strategies to increase the yields of photosynthetic ethanol production. Ruiqing Ni
Results: Even tough the measurements for alcohol concentration were taken with a CO2 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. Ribulose 1,5-bispohsophate 3-Phosphoglycerate Hence we conclude that our constructs are functional. The IPTG inducible promoter for testing purposes in E. coli was pLac (R0011).
RuBisCO + Calvin
Cycle 3-Phosphoglycerate 3x PGM 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. Ethanol Construct: Pyruvate is converted by a Butanol Construct: We expect a broad range of Further research will be done. pyruvate decarboxylase from Z. mobilis to branched-chain higher alcohols, thus we Visit: www.uppsalaigem.org acetaldehyde. A further reaction performed by 2-Phosphoglycerate exemplary demonstrate the mechanism with the alcohol dehydrogenase 2 from S. cerevisiae the pathway for isobutanol production. The leads to ethanol production. Expression of the enzyme alpha-ketoisovalerate decarboxylase construct in Synechocystis sp PCC6803 is ENO from L. lactis sp. converts keto acids to regulated by the copper inducible promoter aldehydes which then are processed to alcohols pPetE. by adh2 from S. cerevisiae. [2]
pPetE T pPetE T
Phosphoenolpyruvate RBS pdc RBS adhII (Y) RBS kivd RBS adhII Isobutanol Ethanol K273019 B0034 K273000 B0034 K273001 B0015 PK K273019 B0034 K273006 B0034 K273005 B0015
Ethanol Module Butanol Module Pdc adh2 adh Pyruvate Aminoac iosynt id PDC B he kivd sis Acetaldehyde 2-keto-isovalerate Isobutanal ENo Enolase PK Pyruvate Kinase CO Krebs CO2 adh Alcohol Dehydrogenase 2 Pdc Pyruvate decarboxylase Cycle Inhibtion: As pyruvate is a precursor of both pathways, we engineered two PGM Phosphoglycerate Mutase di erent approaches to increase the pyruvate levels in order to nally obtain adh2 Alcohol Dehydrogenase 2 more product by redirecting the carbon ux. PDC Pyruvate Dehydrogenase Complex The antisense RNA approach targets the formation of the PDC complex . We kivd Alpha-ketoisovalerate decarboxylase constructed twelve di erent antisense RNA which are expressed under RuBisCO Ribulose-1,5-Bisphosphate Carboxylase Oxygenase copper-inducible promoter pPetE and terminated with T0015. Native Pathway As pirin proteins are known inhibitors of the PDC in other organisms and is Introduced Pathway 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]
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
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