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| Home > Publications database > Metabolic engineering of Pseudomonas taiwanensis for the improved production of styrene |
| Book/Dissertation / PhD Thesis | FZJ-2025-03157 |
2025
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-841-4
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Please use a persistent id in citations: urn:nbn:de:0001-2508051118123.694702301970 doi:10.34734/FZJ-2025-03157
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.
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