Team:Uppsala-Sweden/Butanol

From 2009.igem.org

The Butanol Approach

Our goal is to achieve photosynthetic production of butanol and its derivatives with the cyanobacterium Synechocystis sp. PCC 6803 as the final host organism.

Background

Cyanobacteria have the capability to harvest the energy from the sun and convert it into other forms of energy. The natural way for these organisms is to store it as sugars or other carbohydrates in a way similar to plants. By introducing a cassette of genes for isobutanol production, we would like to derive a cyanobacteria that produces butanol and its derivatives.

The Constructs

We designed two final constructs ( & ) that actually only differ in the promoter, as our final host organism Synechocystis sp PCC6803 does not allow the use of conventional inducible promoters. More information about pPetE, a copper inducible promoter for Synechocystis sp PCC6803, can be found here .

: The Construct for Testing Purposes in E. Coli

: The Final Construct for Synechocystis sp PCC6803

The Pathway

The original pathway to obtain branched-chain higher alcohols was proposed and successfully established in E. coli by Shota Atsumi, Taizo Hanai and James C. Liao from the University of Californa, Los Angeles. [1] As we expect a broad range of higher alcohols, we will exemplary demonstrate the biochemical mechanism with the pathway of isobutanol, the product we expect to be the most abundant.

The precursor for isobutanol production is pyruvate, a central metabolite which is usually derived by the glycolysis. Two pyruvate molecules are converted first to 2-acetolactate and then subsequently to 2,3-dihydroxy-isovalerate. A further dehydration reaction results in our first molecule of interest, 2-ketoisovalerate, which is a precursor for the synthesis of the amino acids valine, alanine and leucine. [2]

So far a native pathway has been utilized, our first BioBrick for the Butanol approach, the codon-optimized version of the alpha-ketoisovalerate decarboxylase (kivd) from Lactococcus lactis ssp. lactis is operating on these products. [3]

This particular enzyme performs a decarboylation reaction, and thus transforms 2-ketoisovalerate to an aldehyde, isobutanal. [4]

Subsequently isobutanal will be oxidized to isobutanol by an enzyme which is encoded by our BioBrick , the alcohol dehydrogenase 2 (adh2) from S. cerevisiae. [5]

Status

as of 2009-10-21

The Butanol construct is completed and is currently being evaluated in E.coli. While the transformation to Synechocystis is done it takes approximately two weeks for the colonies to grow to large enough size to be used in testing.

References

[1] [http://www.ncbi.nlm.nih.gov/pubmed/18172501 Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Atsumi S, Hanai T, Liao JC Nature. 2008 Jan 3;451(7174):86-9]

[2] [http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=VALSYN-PWY MetaCyc Pathway: valine biosynthesis]

[3] [http://www.ncbi.nlm.nih.gov/pubmed/15358422 Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis. de la Plaza M, Fernández de Palencia P, Peláez C, Requena T. FEMS Microbiol Lett. 2004 Sep 15;238(2):367-74]

[4] [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-7643 MetaCyc Reaction: 4.1.1.1]

[5] [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-7657 MetaCyc Reaction: 1.1.1.1]