Team:Bielefeld-CeBiTec/Results/Outlook

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Module III - Isobutanol production

Isobutanol pathway
Building on the results shown in the section Isobutanol pathway further analysis could follow. We could show via SDS Page that potentially all proteins are expressed. Furthermore we could demonstrate that bacteria carrying our constructs produce isobutanol.

Intermediates

Since the cultures carrying the construct BBa_K1465307 produced less isobutanol than cultures carrying the construct BBa_K1465306. It would be interesting to analyze the intermediates of the used pathway (Atsumi et al., 2008). There might be a difference between the amounts of the particular intermediates which could explain the different production rates.

Conditions

Furthermore an increase of the production rate would be an aim of further experiments. Therefore optimized conditions have to be identified. We could show, that the production is increased at a temerature of 30°C. Maybe a temperature between 30°C and 37°C could be tryed out.

Pathway

A further possibility might be to try other genes for the 2-keto-acid pathway, such as the kivD from Lactococcus lactis in our isobutanol production pathway.

Separation

For analysing our isobutanol production we took samples directly of the cultures and analysed the supernatant. The next step would be the separation of isobutanol and the medium respectively the bacteria. The iGEM team NCTU Formosa presented a possible system.

Alcohol dehydrogenase

As described in the section about the alcohol dehydrogenase we could show the successful overexpression of the protein AdhA from L. lactis in E. coli.

Anaerobic cultivation

Further analysis could be an anaerobic cultivation for a complementation analysis of the strain ΔadhE748::kan. Trinh et al., 2011 showed that E. coli cannot grow after the loss of its alcohol dehydrogenase. Complemented with our plasmid (BBa_K1465305) the mutant should grow.


References
  • Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: Nature 451, 86–89.
  • Trinh CT, Li J, Blanch HW, Clark DS., 2011. Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol. In: Appl Environ Microbiol., 77(14): 4894-904

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