Multiple novel membrane proteins involve phthalate ester degradation in Rhodococcus sp. AH-ZY2

Phthalate esters (PAEs) are ubiquitous in the environment, and their microbial metabolism is associated with transport proteins. In this study, multiple novel membrane proteins were identified, revealing their pivotal role in PAEs degradation. Based on whole-genome and transcriptomic analyses, approximately 20 potential PAEs transport membrane proteins were predicted to be involved in PAEs degradation by Rhodococcus sp. AH-ZY2. Among them, four protein genes (0620, 3572, 4497, and 5299) exhibited increased transcription levels (Log₂F(c)>2.8) in response to di-n-octyl-phthalate (DnOP) as the sole carbon source, instead of fructose, were selected for functional verification. Gene knockout and complementation experiments showed that these four transport protein genes, belonging to the MFS family (0620, 3572, and 5299) and the ABC family (4497), respectively, exhibited the function to transport multiple PAEs into cells. Specifically, membrane protein 5299 could transport the most diverse kinds of PAEs. Molecular docking results also showed that Ser and Arg play a key role in the PAEs transport process of membrane protein 5299, and the PAEs-binding pocket size of 5299 in the transition region was larger than that of 0620, 3572, and 4497. Then, the expression of the genes encoding 0620, 3572, 5299, and 4497 was enhanced after they were cloned into the plasmid pNV18 and transformed into the strain AH-ZY2. The enhancement of 5299 expression exhibited a better improvement in PAEs degradation than the other three genes. This study provides four novel transport proteins and a new strategy for effective bioremediation of PAEs via enhanced membrane transport.

Phthalate esters (PAEs) are widely present in the environment, with carcinogenic, teratogenic, and mutagenic toxicity to the human body. The efficient microbial degradation of PAEs is urgent for eco-friendly bioremediation. In addition to PAE esterases, membrane proteins for PAE transport are also important for the microbial degradation of PAEs. However, few experimental reports on the membrane proteins involved in PAE transport, and specifically no studies regarding their underlying transport mechanisms, have been published. Therefore, investigation of the PAE transport mechanisms is crucial for understanding how PAEs enter and exit cells, and it contributes to identifying the rate-limiting steps in PAE degradation. It is conducive to revealing the role of membrane proteins in PAEs degradation, for improving PAE degradation efficiency via membrane protein engineering, or endowing other chassis cells with PAEs degradation capability via constructing membrane protein-esterase co-expressing strains.