Engineering of Pseudomonas taiwanensis VLB120 for the sustainable production of hydroxylated aromatics

Aromatics are valuable compounds with a myriad of applications. They are bulk or fine chemicals, used for the production of e.g. polymers and pharmaceuticals. Consequently, they display an indispensable cornerstone of modern society. The vast majority of them is produced from non-renewable resources in petrochemical processes, implicating many negative aspects associated to the exploitation of oil. An aromatics production based on whole-cell biocatalysis would contribute to a more sustainable bioeconomy. However, the inherent toxicity of many aromatics hampers microbial production and yields achieved in previous studies were mostly low. Due to their robustness and intrinsic protective mechanisms towards aromatics, Pseudomonads display promising hosts for the production of these chemicals. The overall aim of this thesis was to achieve efficient aromatics production from renewable feedstocks using solvent-tolerant Pseudomonas taiwanensis VLB120 as microbial cell factory. The focus was on the production of hydroxylated aromatics derived from de novo-synthesized tyrosine. Phenol was chosen as first target product, as it is an ideal paradigm of a toxic and industrially relevant aromatic. The implementation of a tyrosine phenol-lyase and extensive metabolic engineering of this bacterium, applying a combination of forward and reverse engineering strategies, enabled high-yield phenol production from glucose and glycerol. In case of the latter, the highest reported phenol yield of 18.8% (Cmol/Cmol) was achieved in a mineral medium without the supplementation of any complex additives. The generated tyrosine-overproducing platform strain was further metabolically engineered and adapted to enable 4-vinylphenol production. In this context, extremely high 4 vinylphenol yields of up to 64.6% (Cmol/Cmol) were achieved. This translates into approximately 88% of the theoretical maximum yield. This efficient production came at the cost of very poor growth of the producing strain, hurting the volumetric production rate. To increase productivity and product titers, process engineering should be applied in forthcoming studies. In addition to the metabolic engineering work, streamlined chassis strains of P. taiwanensis VLB120 were generated by the targeted elimination of dispensable and unfavorable cell elements. The large megaplasmid pSTY, proviral segments, and flagella- and biofilm-associated gene clusters were deleted. Thereby, the genome was reduced by up to 10%. The resulting strains showed increased key performance indicators, including enhanced growth rates and biomass yields, likely making them attractive hosts for a broad range of industrial applications. For the process-guided application of custom-tailored strains according to a chassis à la carte principle, we generated streamlined strains only varying concerning their solvent tolerance level resulting from different expression of the solvent efflux pump TtgGHI. To profit from the chassis strains’ superior performance, they were engineered for the production of phenol and 4 vinylphenol, thereby further increasing the titer, yield, and volumetric rate of production. Altogether, this thesis contributed to the fundamental understanding of aromatics metabolic pathways and solvent tolerance in P. taiwanensis, which further strengthens the role of P. taiwanensis VLB120 and the genome-reduced chassis as industrial workhorse

Global control of bacterial nitrogen and carbon metabolism by a PTSNtr-regulated switch

The nitrogen-related phosphotransferase system (PTSNtr) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTSNtr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTSNtr is widely conserved in proteobacteria, highlighting its global importance