Strain and process engineering for the fermentative production of secondary metabolites in Pseudomonas sp. strain VLB120

Due to the foreseeable shortage of fossil resources in the future and the environmental concerns connected to a fossil-based economy, bio-based sustainable processes are more and more in the focus of academic and industrial research. A major bottleneck in this respect is the lack of appropriate whole-cell biocatalysts, tolerant towards relevant reactants and process conditions. This thesis focuses on Pseudomonas sp. strain VLB120, which is known for its intrinsic solvent tolerance towards a variety of toxic chemicals. Using an extensive metabolic engineering approach, the Ehrlich pathway was overexpressed in the desired host. This allowed the production of three different classes of products directly from glucose: isobutanol, isobutyric acid, and (S)-3-hydroxyisobutyric acid. The accumulation of isobutyric acid and (S)-3-hydroxyisobutyric acid was achieved following a genome based random mutagenesis approach with subsequent screening for suitable hosts. The production of isobutanol was naturally limited, due to the kinetically preferred oxidation of its precursor isobutyraldehyde. Using transcriptome analysis responsible aldehyde dehydrogenases were identified and their deletion led to the accumulation of isobutanol. The ability of the strain to form stable biofilms was used in biofilm membrane reactors for the continuous production of 3-hydroxyisobutyric acid. Due to the toxicity of the described compounds, process integration is essential for overall process economics. Thus, in situ separation of isobutanol using gas-stripping, and the abiotic reactive extraction of isobutyric acid using the quaternary ammonium salt Aliquat 336, were investigated and the successful separation of isobutanol during fermentations was demonstrated.