Bioinformatics Analysis and Characterization of Highly Efficient Polyvinyl Alcohol (PVA)-Degrading Enzymes from the Novel PVA Degrader Stenotrophomonas rhizophila QL-P4

Abstract

Polyvinyl alcohol (PVA) is used widely in industry, and associated environmental pollution is a serious problem. Herein, we report a novel, efficient PVA degrader, Stenotrophomonas rhizophila QL-P4, isolated from fallen leaves from a virgin forest in the Qinling Mountains. The complete genome was obtained using single-molecule real-time (SMRT) technology and corrected using Illumina sequencing. Bioinformatics analysis revealed eight PVA/vinyl alcohol oligomer (OVA)-degrading genes. Of these, seven genes were predicted to be involved in the classic intracellular PVA/OVA degradation pathway, and one (BAY15_3292) was identified as a novel PVA oxidase. Five PVA/OVA-degrading enzymes were purified and characterized. One of these, BAY15_1712, a PVA dehydrogenase (PVADH), displayed high catalytic efficiency toward PVA and OVA substrate. All reported PVADHs only have PVA-degrading ability. Most importantly, we discovered a novel PVA oxidase (BAY15_3292) that exhibited higher PVA-degrading efficiency than the reported PVADHs. Further investigation indicated that BAY15_3292 plays a crucial role in PVA degradation in S. rhizophila QL-P4. Knocking out BAY15_3292 resulted in a significant decline in PVA-degrading activity in S. rhizophila QL-P4. Interestingly, we found that BAY15_3292 possesses exocrine activity, which distinguishes it from classic PVADHs. Transparent circle experiments further proved that BAY15_3292 greatly affects extracellular PVA degradation in S. rhizophila QL-P4. The exocrine characteristics of BAY15_3292 facilitate its potential application to PVA bioremediation. In addition, we report three new efficient secondary alcohol dehydrogenases (SADHs) with OVA-degrading ability in S. rhizophila QL-P4; in contrast, only one OVA-degrading SADH was reported previously.IMPORTANCE With the widespread application of PVA in industry, PVA-related environmental pollution is an increasingly serious issue. Because PVA is difficult to degrade, it accumulates in aquatic environments and causes chronic toxicity to aquatic organisms. Biodegradation of PVA, as an economical and environment-friendly method, has attracted much interest. To date, effective and applicable PVA-degrading bacteria/enzymes have not been reported. Herein, we report a new efficient PVA degrader (S. rhizophila QL-P4) that has five PVA/OVA-degrading enzymes with high catalytic efficiency, among which BAY15_1712 is the only reported PVADH with both PVA- and OVA-degrading abilities. Importantly, we discovered a novel PVA oxidase (BAY15_3292) that is not only more efficient than other reported PVA-degrading PVADHs but also has exocrine activity. Overall, our findings provide new insight into PVA-degrading pathways in microorganisms and suggest S. rhizophila QL-P4 and its enzymes have the potential for application to PVA bioremediation to reduce or eliminate PVA-related environmental pollution.

Keywords: PacBio; Stenotrophomonas rhizophila; biodegradation; environmental microorganism; high-throughput sequencing; polyvinyl alcohol; single-molecule real-time; vinyl alcohol oligomers.

FIG 1

Cell growth and polyvinyl alcohol (PVA) degradation curves of Stenotrophomonas rhizophila QL-P4 with initial PVA concentrations of 0.1% (A) and 0.5% (B) as the sole carbon source. The PVA concentration (g/liter) was measured based on a standard curve of PVA (g/liter) versus absorbance at 690 nm (OD₆₉₀). Bacterial growth was measured by the OD₆₀₀ value.

FIG 2

Eight genes in the S. rhizophila QL-P4 genome predicted to participate in PVA degradation. (A) Identification of eight putative PVA-degrading enzymes through domain comparison. The three predicted PVA oxidases, one predicted electron acceptor, one predicted hydrolase, and three predicted vinyl alcohol oligomer (OVA)-degrading enzymes are separately shown as blue, red and orange bold letters on the left. As controls, PVADH/Cytochrome c/OPH from Sphingopyxis sp. 113P3, the annotated PVADH (WP_019184176.1) from Stenotrophomonas maltophilia, and SADH from Thermoanaerobacter ethanolicus are displayed as black bold letters. Domains were searched against the InterPro database (http://www.ebi.ac.uk/interpro/) and are shown as different colored bars. Different domains are color coded and shown at the bottom. Each gray rectangle represents a protein predicted to participate in PVA degradation, and IDs of their corresponding genes are included on the left. Numbers under gray rectangles indicate the relative positions of amino acids counting from the N terminus. (B) Proposed roles of the eight predicted PVA degradation pathway genes. The first step of intracellular PVA degradation is the oxidation of PVA by PVADHs (BAY15_2325/1712/3292) with cytochrome c as an electron acceptor (BAY15_0291). The second step is the hydrolysis of the β-diketone of oxiPVA by OPH or BPH (BAY15_0160) to produce a methyl ketone and an aldehyde. OVA, another product of PVA degradation, is intracellularly degraded by SADHs (BAY15_3143/3123/0976).

FIG 3

PVA degradation curves of S. rhizophila QL-P4 with and without BAY15_3292 in the presence of 0.1% PVA as the sole carbon source.

FIG 4

SDS-PAGE identification of the secretion of BAY15_3292 by 2D-LC-MS/MS. (A) Secreted S. rhizophila QL-P4 proteins in medium containing 0.1% PVA. Lanes: marker, protein markers; BAY15_3292, purified BAY15_3292; secreted proteins, S. rhizophila QL-P4 secreted proteins. (B) 2D-LC-MS/MS identification of BAY15_3292 among proteins secreted from S. rhizophila QL-P4. The two representative spectra show the b/y ions used for identification of unique BAY15_3292 peptides.