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bioRxiv · 10.1101/2023.09.27.559296

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater

Yu, Y.·Trottmann, N. F.·Schaerer, M. R.·Fenner, K.·Robinson, S. L.
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Abstract

Organic contaminants enter aquatic ecosystems from various sources, including wastewater treatment plant effluent. Freshwater biofilms play a major role in the removal of organic contaminants from receiving water bodies, but knowledge of the molecular mechanisms driving contaminant biotransformations in complex stream biofilm (periphyton) communities remains limited. Previously, we demonstrated that biofilms in experimental flume systems grown at higher ratios of treated wastewater (WW) to stream water displayed an increased biotransformation potential for a number of organic contaminants. We identified a positive correlation between WW percentage and biofilm biotransformation rates for the widely-used insect repellent, N,N-diethyl-meta-toluamide (DEET). Here, we conducted deep shotgun sequencing of flume biofilms and identified a positive correlation between WW percentage and metagenomic read abundances of DEET hydrolase (DH) homologs. To test the causality of this association, we constructed a targeted metagenomic library of DH homologs from flume biofilms. We screened our complete metagenomic library for activity with four different substrates and a subset thereof with 183 WW-related organic compounds. The majority of active hydrolases in our library preferred aliphatic and aromatic ester substrates while, remarkably, only a single reference enzyme was capable of DEET hydrolysis. Of the 626 total enzyme-substrate combinations tested, approximately 5% were active enzyme-substrate pairs. Metagenomic DH family homologs revealed a broad substrate promiscuity spanning 22 different compounds when summed across all enzymes tested. We biochemically characterized the most promiscuous and active enzymes identified based on metagenomic analysis from uncultivated Rhodospirillaceae and Planctomycetaceae. In addition to characterizing new DH family enzymes, we exemplified a framework for linking metagenome-guided hypothesis generation with experimental validation. Overall, this study expands the scope of known enzymatic contaminant biotransformations for metagenomic hydrolases from WW-receiving stream biofilm communities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/559296v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b5cbaforg.highwire.dtl.DTLVardef@4f73f7org.highwire.dtl.DTLVardef@467d16org.highwire.dtl.DTLVardef@1be598d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMetagenomic DEET hydrolase abundances higher in biofilms grown in treated wastewater. C_LIO_LIEleven out of 64 metagenomic hydrolases tested exhibited hydrolysis activity. C_LIO_LIRelated enzymes in a single family of DEET hydrolases biotransform 20+ contaminants. C_LIO_LIReference DEET hydrolase shows a substrate preference for benzamide moieties. C_LIO_LI True DEET hydrolases are in low abundance even in biofilms that degrade DEET. C_LI

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Yu, Y., Trottmann, N. F., Schaerer, M. R., Fenner, K., Robinson, S. L.. 2023-09-28. Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater. https://doi.org/10.1101/2023.09.27.559296

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