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Aromatics are versatile compounds required for numerous applications used in everyday life. Today, most aromatics are produced from crude oil in petrochemical processes, which goes along with fossil resource depletion and negative environmental impacts. Microbial catalysis can offer alternative production concepts for aromatics, in processes that utilize renewable substrates and cause less emissions. As many aromatics display enhanced toxicity towards microbes, the utilization of especially robust host cells is required to allow their efficient microbial production. Bacteria of the genus Pseudomonas thrive in challenging environments and possess the native ability to cope with many different chemicals and are thus promising candidates for the production of toxic aromatics. The aim of this thesis was the domestication of Pseudomonas species to produce various aromatic chemicals. For this, the aromatic amino acid L-phenylalanine was defined as inherent precursor. The application of rational metabolic engineering strategies, including the deletion and alteration of chromosomal elements and the overexpression of heterologous genes, resulted in Pseudomonas taiwanensis chassis strains that efficiently accumulate L-phenylalanine. The expression of a phenylalanine ammonia lyase from the plant Arabidopsis thaliana then allowed synthesis of the aromatic building block trans-cinnamate with yields of up to 48% Cmol Cmol-1using glycerol as sole carbon source. These strains were then used as platform to produce several chemical compounds of industrial interest. A ferulic acid decarboxylase led to the production of styrene, the monomer of polystyrene, which is used in different plastic products. In another approach, the plantpolyphenol pinosylvin was produced using the t-cinnamate-producing chassis strains by additional expression of a 4-coumarate ligase from Streptomyces coelicolor and a stilbene synthase from Arachis hypogaea. This work further describes the establishment of a novel pathway for the production of benzoate via enzymes of the phenylpropanoid degradation pathway of Corynebacterium glutamicum, which also allowed the production catechol and cis,cismuconatevia native pathways of Pseudomonas. In addition, a novel tool for the targeted integration of heterologous genetic elements into ribosomal operons of Pseudomonas was established. Here, the high activity of ribosomal promoters in combination with transcript-modulation by synthetic elements allowed high expression rates of foreign genes. The tool furthermore allowed analysis of the different ribosomal operons in the strain Pseudomonas putida S12. In summary, this thesis describes a rational approach towards the development of sustainable processes for the production of a variety of L-phenylalanine derived chemicals. The novel tool for heterologous gene expression delivers a further engineering approach of highly versatile Pseudomonas species, thereby bringing the application of this interesting host one step closer towards efficient industrial processes.

Dissertation, RWTH Aachen University, 2020; Aachen : Apprimus Verlag, Applied microbiology 21, 1 Online-Ressource (XIII, 133 Seiten) : Illustrationen (2020). = Dissertation, RWTH Aachen University, 2020, Aromatics are versatile compounds required for numerous applications used in everyday life. Today, most aromatics are produced from crude oil in petrochemical processes, which goes along with fossil resource depletion and negative environmental impacts. Microbial catalysis can offer alternative production concepts for aromatics, in processes that utilize renewable substrates and cause less emissions. As many aromatics display enhanced toxicity towards microbes, the utilization of especially robust host cells is required to allow their efficient microbial production. Bacteria of the genus Pseudomonas thrive in challenging environments and possess the native ability to cope with many different chemicals and are thus promising candidates for the production of toxic aromatics. The aim of this thesis was the domestication of Pseudomonas species to produce various aromatic chemicals. For this, the aromatic amino acid L-phenylalanine was defined as inherent precursor. The application of rational metabolic engineering strategies, including the deletion and alteration of chromosomal elements and the overexpression of heterologous genes, resulted in Pseudomonas taiwanensis chassis strains that efficiently accumulate L-phenylalanine. The expression of a phenylalanine ammonia lyase from the plant Arabidopsis thaliana then allowed synthesis of the aromatic building block trans-cinnamate with yields of up to 48% Cmol Cmol-1using glycerol as sole carbon source. These strains were then used as platform to produce several chemical compounds of industrial interest. A ferulic acid decarboxylase led to the production of styrene, the monomer of polystyrene, which is used in different plastic products. In another approach, the plantpolyphenol pinosylvin was produced using the t-cinnamate-producing chassis strains by additional expression of a 4-coumarate ligase from Streptomyces coelicolor and a stilbene synthase from Arachis hypogaea. This work further describes the establishment of a novel pathway for the production of benzoate via enzymes of the phenylpropanoid degradation pathway of Corynebacterium glutamicum, which also allowed the production catechol and cis,cismuconatevia native pathways of Pseudomonas. In addition, a novel tool for the targeted integration of heterologous genetic elements into ribosomal operons of Pseudomonas was established. Here, the high activity of ribosomal promoters in combination with transcript-modulation by synthetic elements allowed high expression rates of foreign genes. The tool furthermore allowed analysis of the different ribosomal operons in the strain Pseudomonas putida S12. In summary, this thesis describes a rational approach towards the development of sustainable processes for the production of a variety of L-phenylalanine derived chemicals. The novel tool for heterologous gene expression delivers a further engineering approach of highly versatile Pseudomonas species, thereby bringing the application of this interesting host one step closer towards efficient industrial processes., Published by Apprimus Verlag, Aachen

Frontiers in Bioengineering and Biotechnology 7, 312 (2019). doi:10.3389/fbioe.2019.00312, Published by Frontiers Media, Lausanne

Frontiers in Bioengineering and Biotechnology 7, 130 (2019). doi:10.3389/fbioe.2019.00130, Published by Frontiers Media, Lausanne

Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium Pseudomonas taiwanensis VLB120 towards accumulation of L-phenylalanine and its conversion into the chemical building block t cinnamate. We recently reported rational engineering of Pseudomonas towards L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t-cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana. The engineered strains produced t cinnamate with yields of 23% and 39% Cmol Cmol-1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol-1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t-cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 towards t-cinnamate.