Engineering of acetogens for fuel production using H2 and CO2

Background

The transition towards a bio-based economy demands the development of fermentation-based processes, which are economically competitive with the currently employed unsustainable production processes. Unfortunately, only very few natural organisms are suitable for their direct application in an industrial process. Therefore, efficient metabolic engineering via targeted genome engineering is required and the development and use of genome engineering tools generally applicable to many model and non-model organisms is of great importance. Anaerobic acetogenic microorganisms use the Wood-Ljungdahl pathway to reduce CO₂ with H₂ to acetate, ethanol and other products.

Aim

Clostridium autoethanogenum is our model acetogen and converts H₂ and CO₂ into mainly acetate. We have established a conjugation protocol and a CRISPR-Cas-based engineering tool for this organism. Now, in the final stage of the project we aim to knockout a few genes involved in acetyl-CoA conversion and force the organism to make more ethanol instead of acetate. Secondly, we want to heterologously express, purify and characterize several alcohol dehydrogenases from the same organism and identify their cofactor specificity.

Techniques

Multiple techniques are used to achieve our targets including CRISPR-Cas genome editing, anaerobic culturing, HPLC, GC, FPLC and regular plasmid design and cloning.