New photosynthetic cell factories for bio-butanol production

In this project we:
1. Assemble a number of butanol fermentation pathways (for the production of 1-, 2- and/or iso-butanol) in Escherichia coli. These pathways will be composed of enzymes derived from oxygen tolerant micro-organisms, like lactic acid bacteria, enterobacteria and/or yeast strains;
2. Optimize the functioning of these pathways in a dedicated E. coli strain lacking all three terminal oxidases;
3. Heterologously express the best of them in the cyanobacterium Synechocystis sp. PCC6803 under control of suitable induction systems; and
4. Optimize butanol production from CO2 in Synechocystis sp. PCC6803. This approach in synthetic biology will provide new tools for the sustainable production of a key innovative biofuel: butanol.

Key scientific challenges in this project are (i) Identification of very oxygen tolerant versions of the range of fermentative enzymes that will be needed to construct the three butanol fermentation pathways that will be constructed; (ii) Optimization of pathway function of sets of these enzymes in the intracellular milieu; which can be achieved e.g. with enzyme evolution techniques; and (iii) Decreasing product sensitivity of cyanobacteria for the three butanol isomers to the level that an economically viable production system can be designed. As this product sensitivity presumably is a multi-factorial trait of the cyanobacteria, here directed evolution experiments, complemented by directed engineering steps, will lead the way. The directed evolution can be driven by exposure to gradually increasing concentrations of the biofuel product. The directed engineering will focus on the heterologous expression of hydrocarbon efflux systems.

The team that will carry out this project is composed of tenured and temporary staff members of the Molecular Microbial Physiology Research Group of the University of Amsterdam.

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