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de Winde, J.

Publications and source records attributed to de Winde, J..

3 recordsLinked to original sources

Genome Mining and Pangenome Analysis of the Stutzerimonas Genus: a Novel Source of Plastic-Degrading Enzymes

Nowadays, finding new sustainable ways to combat plastic pollution is a pressing challenge. Here, we provide a comprehensive genome mining analysis of 284 publicly available Stutzerimonas genomes for potential PET-active enzymes (PETases). While Stutzerimonas is a relatively newly established genus, it emerges as an interesting candidate in the search for novel biocatalysts. Hence, the first pangenome assessment of this genus based on its high-quality publicly available genomes was performed. An increasingly open pangenome was revealed, suggesting the versatility and adaptability of these strains to a variety of ecological niches. Moreover, functional characterisation of a new isolate, Stutzerimonas frequens VG-9, was carried out, confirming that enzymes found via in silico analyses may indeed display activity towards different polyesters. In summary, this study provides insights into the diversity of PETase homologues within still underexplored bacterial hosts, offering new perspectives for enzyme discovery in the Pseudomonadaceae family. Impact StatementMicrobial enzymes known as PETases have emerged as promising candidates for the biological degradation of PET. This study investigated the potential of underexplored bacterial genera by genome mining of PETase homologues. Our findings provide new insights into the distribution of PETase-like enzymes in the Pseudomonadaceae family, offering a more comprehensive view of their plastic degradation capacity. These results hold practical implications for the development of optimized enzyme discovery strategies, while also highlighting the vast genetic plasticity of Pseudomonadaceae. We also provided the first report on the Stutzerimonas pangenome and insights into the enzymatic activity towards polyesters of a newly isolated strain. Hence, the role of this genus as a highly adaptable and versatile entity was reinforced, further disclosing it as a potential source of novel biocatalysts. Data SummaryThe genome of S. frequens VG9 has been deposited in Genbank under the accession number SAMN49487720. The accession numbers of all analyzed genomes are listed in Tables S2 and S3 (available in the online Supplementary Material).

microbiology↗

Lignin degradation and valorization by Pseudomonas putida KT2440: a new role for glutathione peroxidase

Lignin, a complex natural aromatic polymer, poses significant challenges to its efficient degradation, hindering the utilization of biomass for many industrial applications. Bacterial degradation of lignin may offer a promising solution to this challenge. This project aimed at elucidating the function of secreted oxidative enzymes from Pseudomonas putida involved in lignin degradation and utilization. Using CRISPR-Cas9 and CRISPR-Cas3 systems, the putative lignin-degrading versatile peroxidase gene (VP; PP_1686, originally annotated as glutathione peroxidase GPx) and dye-decolorizing peroxidase gene (PP_3248) were individually knocked out from P. putida KT2440. The {Delta}PP_1686 mutant exhibited impaired growth and utilization of lignin-derived compounds. This correlated with reduced expression of p-hydroxybenzoate hydroxylase pobA and of DNA repair modules, alongside compensatory upregulation of energy and redox supply pathways. This work expands our knowledge on bacterial glutathione peroxidase by presenting a role beyond ROS scavenging. This work revealed the importance of P. putida VP/GPx in maintaining redox balance while supporting lignin-derived aromatic metabolism, offering new targets for future investigation into stress-metabolism crosstalk and lignin valorization strategies.

microbiology↗

Finding needles in haystacks: identification of novel conserved PETase enzymes in Streptomyces

The rising use of plastic results in an appalling amount of waste which scatters into the environment affecting environmental, animal, and human health. One of these plastics is PET which is mainly used for bottles and textiles. In this research, we investigate the PET degrading ability of the IsPETase homolog ScLipA from Streptomyces coelicolor. Of 96 different Streptomyces strains screened, 18 % were able to degrade the model substrate BHET. Three different variants of lipase A, named ScLipA, S2LipA and S92LipA were identified and analyzed in detail. The lipA gene was deleted from S. coelicolor M145 using CRISPR/Cas9, resulting in reduced BHET degradation. LipA overexpression in the knock-out background significantly enhanced BHET degradation. All three enzymes were expressed in E. coli BL21 for protein purification and biochemical analysis, showing that enzymatic activity most likely resides in a dimeric form of the enzyme. The optimum pH and temperature were determined to be pH 7 and 25 {degrees}C for all three variants. Using these conditions, the activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and small indents on the surface of PET films, consistent with PET-degrading activity. The frequent occurrence of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more hydrophobic substrates such as these polluting plastics in the environment.

microbiology↗