Biochemical and Multi-Omics Approaches To Obtain Molecular Insights into the Catabolism of the Plasticizer Benzyl Butyl Phthalate in Rhodococcus sp. Strain PAE-6

Phthalate diesters are extensively used as plasticizers in manufacturing plastic materials; however, because of their estrogenic properties, these chemicals have emerged as a global threat to human health. The present study investigated the course of degradation of a widely used plasticizer, benzyl butyl phthalate (BBP), by the bacterium PAE-6, belonging to the genus Rhodococcus. The metabolism of BBP, possessing structurally dissimilar side chains, was evaluated biochemically using a combination of respirometric, chromatographic, enzymatic, and mass-spectrometric analyses, depicting pathways of degradation. Consequently, the biochemical observations were corroborated by identifying possible catabolic genes from whole-genome analysis, and the involvement of inducible specific esterases and other degradative enzymes was validated by transcriptomic, reverse transcription-quantitative PCR (RT-qPCR) and proteomic analyses. Nonetheless, phthalic acid (PA), an intermediate of BBP, could not be efficiently metabolized by strain PAE-6, although the genome contains a PA-degrading gene cluster. This deficiency of complete degradation of BBP by strain PAE-6 was effectively managed by using a coculture of strains PAE-6 and PAE-2. The latter was identified as a Paenarthrobacter strain which can efficiently utilize PA. Based on sequence analysis of the PA-degrading gene cluster in strain PAE-6, it appeared that the alpha subunit of the multicomponent phthalate 3,4-dioxygenase harbors a number of altered residues in the multiple sequence alignment of homologous subunits, which may play a role(s) in poor turnover of PA. IMPORTANCE Benzyl butyl phthalate (BBP), an estrogenic, high-molecular-weight phthalic acid diester, is an extensively used plasticizer throughout the world. Due to its structural rigidity and hydrophobic nature, BBP gets adsorbed on sediments and largely escapes the biotic and abiotic degradative processes of the ecosystem. In the present study, a potent BBP-degrading bacterial strain belonging to the genus Rhodococcus was isolated that can also assimilate a number of other phthalate diesters of environmental concern. Various biochemical and multi-omics analyses revealed that the strain harbors all the required catabolic machinery for the degradation of the plasticizer and elucidated the inducible regulation of the associated catabolic genes and gene clusters.