TY  - THES
AU  - Lechtenberg, Thorsten
TI  - Tolerance engineering of Pseudomonas for the efficient conversion and production of aldehydes
VL  - 292
PB  - Düsseldorf
VL  - Dissertation
CY  - Jülich
M1  - FZJ-2024-07180
SN  - 978-3-95806-817-9
T2  - Schriften des Forschungszentrums Jülich Reihe Schlüsseltechnologien / Key Technologies
SP  - XVI, 185
PY  - 2025
N1  - Dissertation, Düsseldorf, 2024
AB  - Biocatalysis holds promise to tackle the sustainability challenges faced by chemical industry due to climate change and depletion of fossil resources. However, obstacles emerge regarding the compatibility of several important chemicals, notably aldehydes, with biological systems, even if remarkably robust workhorses such as bacteria of the Pseudomonas clade are employed. This is related to the high and versatile reactivity of aldehydes, which is both their greatest asset and the root cause of their toxicity. Competitive biocatalytic processes involving these substances thus require tolerance-improved host organisms. In view of the constantly growing demand for renewable and ecologically produced plastics, the biocatalytic oxidation of the burgeoning platform chemical 5-(hydroxymethyl)furfural (HMF) to 2,5-furandicarboxylic acid (FDCA) is of particular interest since FDCA can substitute structurally similar and fossilbased terephthalic acid in polyesters. With the periplasmic oxidoreductase complex PaoEFG and the cytoplasmic dehydrogenases AldB-I and AldB-II, the primary enzymes responsible for the oxidation of HMF and further aromatic aldehydes like 4-hydroxybenzaldehyde and vanillin by P. taiwanensis VLB120 and P. putida KT2440 were uncovered. This marks a significant advancement from former black-box application of these strains to specialized biocatalysts with fine-tuned properties. To illustrate, overexpression of the newly characterized genes resulted in so-called BOX-strains (Boosted OXidation) with up to tenfold increased initial oxidation rates in comparison to the wild type. As a result, the new variants exhibited increased robustness when growing in presence of HMF and also proved to be more efficient for the complete oxidation of the aldehyde to the industrial target compound FDCA. Furthermore, tolerance mechanisms distinct from rapid oxidation were sought applying an adaptive laboratory evolution approach. A ROX (Reduced OXidation) deletion mutant with diminished aldehyde conversion ability was subjected to steady HMF stress. This yielded toleranceimproved strains through the unforeseen inactivation of the regulator MexT and the associated shutdown of the efflux pump MexEF-OprN. Another potential use for oxidation-deficient, yet solvent-tolerant, Pseudomonads is the biosynthesis of aromatic aldehydes, as showcased with the popular aroma compound t-cinnamaldehyde. In conclusion, this thesis contributes to the fundamental understanding of aromatic aldehyde conversion by P. taiwanensis VLB120 and P. putida KT2440 by unveiling the underlying enzymes which were shown to constitute the organisms’ main tolerance mechanism against these toxic substances. Their overexpression in BOX strains strongly increases aldehyde tolerance, and enables improved FDCA production by boosted HMF oxidation. Reduced aldehyde oxidation and reduction (ROAR) unlocks P. taiwanensis VLB120 for the (de novo) production of valuable aromatic aldehydes or aldehyde-derived products, thereby expanding the product portfolio of this aspiring microbial cell factory.
LB  - PUB:(DE-HGF)3 ; PUB:(DE-HGF)11
DO  - DOI:10.34734/FZJ-2024-07180
UR  - https://juser.fz-juelich.de/record/1034398
ER  -