Project description:The Halopseudomonas species, formerly classified as Pseudomonas pertucinogena lineage, form a unique phylogenetic branch within the Pseudomonads. Most strains have recently been isolated from challenging habitats including oil- or metal-polluted sites, deep sea, and intertidal zones, suggesting innate resilience to physical and chemical stresses. Despite their comparably small genomes, these bacteria synthesise several biomolecules with biotechnological potential and a role in the degradation of anthropogenic pollutants has been suggested for some Halopseudomonads. Until now, these bacteria are not readily amenable to existing cultivation and cloning methods. We addressed these limitations by selecting four Halopseudomonas strains of particular interest, namely H. aestusnigri, H. bauzanensis, H. litoralis, and H. oceani to establish microbiological and molecular genetic methods. We found that C4 -C10 dicarboxylic acids serve as viable carbon sources in both complex and mineral salt cultivation media. We also developed plasmid DNA transfer protocols and assessed vectors with different origins of replication and promoters inducible with isopropyl-β-d-thiogalactopyranoside, l-arabinose, and salicylate. Furthermore, we have demonstrated the simultaneous genomic integration of expression cassettes into one and two attTn7 integration sites. Our results provide a valuable toolbox for constructing robust chassis strains and highlight the biotechnological potential of Halopseudomonas strains.
Project description:Impranil® DLN-SD is a poly(ester-urethane) (PEU) that is widely used as coating material for textiles to fine-tune and improve their properties. Since coatings increase the complexity of such plastic materials, they can pose a hindrance for sustainable end-of-life solutions of plastics using enzymes or microorganisms. In this study, we isolated Halopseudomonas formosensis FZJ due to its ability to grow on Impranil DLN-SD and other PEUs as sole carbon sources. The isolated strain was exceptionally thermotolerant as it could degrade Impranil DLN-SD at up to 50°C. We identified several putative extracellular hydrolases of which the polyester hydrolase Hfor_PE-H showed substrate degradation of Impranil DLN-SD and thus was purified and characterized in detail. Hfor_PE-H showed moderate temperature stability (Tm = 53.9°C) and exhibited activity towards Impranil DLN-SD as well as polyethylene terephthalate. Moreover, we revealed the enzymatic release of monomers from Impranil DLN-SD by Hfor_PE-H using GC-ToF-MS and could decipher the associated metabolic pathways in H. formosensis FZJ. Overall, this study provides detailed insights into the microbial and enzymatic degradation of PEU coatings, thereby deepening our understanding of microbial coating degradation in both contained and natural environments. Moreover, the study highlights the relevance of the genus Halopseudomonas and especially the novel isolate and its enzymes for future bio-upcycling processes of coated plastic materials.