Assessment of New and Genome-Reduced Pseudomonas Strains Regarding Their Robustness as Chassis in Biotechnological Applications.

Organic olvent-tolerant strains of the Gram-negative bacterial genus Pseudomonas are discussed as potential biocatalysts for the biotechnological production of various chemicals. However, many current strains with the highest tolerance are belonging to the species P. putida and are classified as biosafety level 2 strains, which makes them uninteresting for the biotechnological industry. Therefore, it is necessary to identify other biosafety level 1 Pseudomonas strains with high tolerance towards solvents and other forms of stress, which are suitable for establishing production platforms of biotechnological processes. In order to exploit the native potential of Pseudomonas as a microbial cell factory, the biosafety level 1 strain P. taiwanensis VLB120 and its genome-reduced chassis (GRC) variants as well as the plastic-degrading strain P. capeferrum TDA1 were assessed regarding their tolerance towards different n-alkanols (1-butanol, 1-hexanol, 1-octanol, 1-decanol). Toxicity of the solvents was investigated by their effects on bacterial growth rates given as the EC50 concentrations. Hereby, both toxicities as well as the adaptive responses of P. taiwanensis GRC3 and P. capeferrum TDA1 showed EC50 values up to two-fold higher than those previously detected for P. putida DOT-T1E (biosafety level 2), one of the best described solvent-tolerant bacteria. Furthermore, in two-phase solvent systems, all the evaluated strains were adapted to 1-decanol as a second organic phase (i.e., OD560 was at least 0.5 after 24 h of incubation with 1% (v/v) 1-decanol), which shows the potential use of these strains as platforms for the bio-production of a wide variety of chemicals at industrial level.

An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene.

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

Toward Biorecycling: Isolation of a Soil Bacterium That Grows on a Polyurethane Oligomer and Monomer.

The fate of plastic waste and a sustainable use of synthetic polymers is one of the major challenges of the twenty first century. Waste valorization strategies can contribute to the solution of this problem. Besides chemical recycling, biological degradation could be a promising tool. Among the high diversity of synthetic polymers, polyurethanes are widely used as foams and insulation materials. In order to examine bacterial biodegradability of polyurethanes, a soil bacterium was isolated from a site rich in brittle plastic waste. The strain, identified as Pseudomonas sp. by 16S rRNA gene sequencing and membrane fatty acid profile, was able to grow on a PU-diol solution, a polyurethane oligomer, as the sole source of carbon and energy. In addition, the strain was able to use 2,4-diaminotoluene, a common precursor and putative degradation intermediate of polyurethanes, respectively, as sole source of energy, carbon, and nitrogen. Whole genome sequencing of the strain revealed the presence of numerus catabolic genes for aromatic compounds. Growth on potential intermediates of 2,4-diaminotoluene degradation, other aromatic growth substrates and a comparison with a protein data base of oxygenases present in the genome, led to the proposal of a degradation pathway.