% IMPORTANT: The following is UTF-8 encoded.  This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.

@PHDTHESIS{Rnitz:1044386,
      author       = {Rönitz, Jakob},
      title        = {{M}etabolic engineering of {P}seudomonas taiwanensis for
                      the improved production of styrene},
      volume       = {297},
      school       = {Düsseldorf},
      type         = {Dissertation},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2025-03157},
      isbn         = {978-3-95806-841-4},
      series       = {Schriften des Forschungszentrums Jülich Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {XII, 147},
      year         = {2025},
      note         = {Dissertation, Düsseldorf, 2025},
      abstract     = {Styrene is an important building block for polymers and
                      that is found in a broad spectrum of products that are
                      omnipresent in our modern society, including consumer
                      electronics, toys, packaging material, and car tires.
                      However, the production of styrene is currently solely based
                      on the petrochemical industry, which does not represent a
                      viable long-term strategy due to the limited abundance of
                      fossil resources. Biotechnological production of this
                      compound provides a sustainable alternative to the supply
                      issue, but the high toxicity and volatility of styrene poses
                      challenges for the development of a bioprocesses. Due to
                      high product toxicity, the use of a solvent-tolerant host
                      organism such as Pseudomonas taiwanensis VLB120 is
                      favourable for this application. However, cultivation of
                      bacteria in presence of volatile solvents also poses special
                      requirements to the cultivation system, resulting in limited
                      throughput and high manual workload for experiments. In this
                      thesis, this challenge was addressed by the development of
                      the SIGHT (solvent-tight incubation and growth monitoring in
                      high throughput) system, which prevents evaporation of
                      volatile compounds and allows to utilise the Growth Profiler
                      (EnzyScreen) platform, enabling incubation and non-invasive
                      online growth monitoring of up to 240 cultures in parallel.
                      The high-throughput capacity of the SIGHT system was
                      demonstrated by adaptive laboratory evolution (ALE) of P.
                      taiwanensis GRC3 to further increase styrene tolerance,
                      which allowed isolation of clones with improved growth in
                      presence of a second phase of styrene. Furthermore, a
                      solvent-inducible biosensor was applied to determine styrene
                      concentrations in the cytosol of a solvent-sensitive and
                      solventtolerant P. taiwanensis strain at different levels of
                      exposure, which enabled calculation of styrene accumulation
                      in the inner membrane. When exposed to a second phase of
                      styrene, the concentration in the cytosol of
                      solvent-tolerant P. taiwanensis GRC2 was only 0.45 mM, which
                      is 6.2-fold lower compared to the medium, due to activity of
                      the TtgGHI solvent efflux pump harboured by this strain.
                      This allowed to gain new insights into the physiology of
                      solvent-tolerant Pseudomonads under stress conditions and
                      further highlighted their suitability as hosts for styrene
                      bioproduction. However, maintaining activity of the TtgGHI
                      solvent efflux pump is highly energy demanding and puts an
                      additional burden on the metabolism of styrene production
                      strains, which also need to overproduce L-phenylalanine, the
                      precursor required for styrene biosynthesis. The styrene
                      biosynthesis pathway consisting of a phenylalanine
                      ammonia-lyase (PAL) from Arabidopsis thaliana and ferulic
                      acid decarboxylase (FDC) from Saccharomyces cerevisiae was
                      integrated into the genome of L-phenylalanine-overproducing
                      strain P. taiwanensis GRC3 Δ8ΔpykA-tap and genetic
                      engineering was applied to balance precursor biosynthesis
                      and strain fitness. This optimisation increased de novo
                      production of styrene from glucose to a concentration of
                      1.30 mM in the aqueous phase (2.68 mM in total, including
                      gas phase), representing an improvement of $8.1\%$ compared
                      to the starting strain. Furthermore, highly styrene-tolerant
                      P. taiwanensis strains without modified L-phenylalanine
                      biosynthesis were applied for biotransformation of
                      t-cinnamate to styrene as an alternative to de novo
                      biosynthesis. This approach allowed complete conversion of
                      up to 50 mM t-cinnamate, which corresponds to saturation of
                      the medium and formation of a second phase of styrene in the
                      culture. Overall, this thesis contributed to physiological
                      understanding of solvent tolerance in P. taiwanensis as well
                      as the balance between tolerance and styrene biosynthesis,
                      which facilitates the suitability of this host organism for
                      further development of a sustainable styrene production
                      process.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2171 - Biological and environmental resources for
                      sustainable use (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2171},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2508051118123.694702301970},
      doi          = {10.34734/FZJ-2025-03157},
      url          = {https://juser.fz-juelich.de/record/1044386},
}