Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

Since high-value bacterial secondary metabolites, including antibiotics, are often naturally produced in only low amounts, their efficient biosynthesis typically requires the transfer of entire metabolic pathways into suitable bacterial hosts like Pseudomonas putida. Stable maintenance and sufficient expression of heterologous pathway-encoding genes in host microbes, however, still remain key challenges. In this study, the 21 kb prodigiosin gene cluster from Serratia marcescens was used as a reporter to identify genomic sites in P. putida KT2440 especially suitable for maintenance and expression of pathway genes. After generation of a strain library by random Tn5 transposon-based chromosomal integration of the cluster, 50 strains exhibited strong prodigiosin production. Remarkably, chromosomal integration sites were exclusively identified in the seven rRNA-encoding rrn operons of P. putida. We could further demonstrate that prodigiosin production was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted as well as (ii) the distance between the rrn promoter and the inserted prodigiosin biosynthetic genes. In addition, the recombinant strains showed high stability upon subculturing for many generations. Consequently, our findings demonstrate the general applicability of rDNA loci as chromosomal integration sites for gene cluster expression and recombinant pathway implementation in P. putida KT2440.

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 (T _m = 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.

Numerous strategies for the biodegradation and upcycling of polyethylene terephthalate (PET) are under investigation. Here, we present a proof-of-concept study for reprogramming the Escherichia coli BL21(DE3) strain to degrade PET nanoparticles (nPET) without introducing foreign DNA and compromising native cellular fitness. In brief, native proteins selected in silico from the genome were repurposed to acquire artificial PETase activity without compromising their function and were subsequently replaced via CRISPR/Cas9 editing. A variant of the transport protein LsrB, selected for its ability to bind PET, was engineered to degrade PET powder (at 37-60°C). Building on LsrB periplasmic localization, we engineered a strain that degrades nPET at 37°C. The strain was further engineered to grow on nPET degradation products and produce valuable compounds. Our method, which is applicable across diverse genomes and microbial chassis, expands the potential of metabolic engineering to address plastic biodegradation and upcycling while reducing reliance on foreign DNA.

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 C ₄ ‐C ₁₀ 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 att Tn7 integration sites. Our results provide a valuable toolbox for constructing robust chassis strains and highlight the biotechnological potential of Halopseudomonas strains.

Many species of the genus Pseudomonas are known to be highly tolerant to solvents and other environmental stressors. Based on phylogenomic and comparative genomic analyses, several Pseudomonas species were recently transferred to a new genus named Halopseudomonas. Because of their unique enzymatic machinery, these strains are being discussed as novel biocatalysts in biotechnology. In order to test their growth parameters and stress tolerance, five Halopseudomonas strains were assessed regarding their tolerance toward different n-alkanols (1-butanol, 1-hexanol, 1-octanol, 1-decanol), as well as to salt stress and elevated temperatures. The toxicity of the solvents was investigated by their effects on bacterial growth rates and presented as EC50 concentrations. Hereby, all Halopseudomonas strains showed EC50 values up to two-fold lower than those previously detected for Pseudomonas putida. In addition, the activity of the cis-trans isomerase of unsaturated fatty acids (Cti), which is an urgent stress response mechanism known to be present in all Pseudomonas species, was monitored in the five Halopseudomonas strains. Although several of the tested species were known to contain the cti gene, no significant phenotypic activity could be detected in the presence of the assayed stressors. A bioinformatic analysis of eight cti-carrying Halopseudomonas strains examining promotor binding sites, binding motifs and signal peptides showed that most of the cti genes have a lipoprotein signal peptide and promotor regions and binding motifs that do not coincide with those of Pseudomonas. These insights represent putative reasons for the absence of the expected Cti activity in Halopseudomonas, which in turn has always been observed in cti-carrying Pseudomonas. The lack of Cti activity under membrane stress conditions when the cti gene is present has never been documented, and this could represent potential negative implications on the utility of the genus Halopseudomonas for some biotechnological applications.