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Biology subjects

Chowreddy, R. R.

Publications and source records attributed to Chowreddy, R. R..

4 recordsLinked to original sources

Biodegradation of components from an oxidized polyethylene by a Rhodococcus strain isolated from the gut of Atlantic Salmon

Polyethylene (PE) is the most produced synthetic polymer and as a result, a major source of microplastic waste accumulating globally. Exposure to photo- and thermo-oxidative conditions in the environment can cause PE to degrade into carbonyl-containing compounds, hydrocarbons, and low molecular weight PE (LMWPE). In both marine and freshwater ecosystems, fish, including Atlantic salmon, can ingest PE and its derivatives, creating opportunities for interactions with their gut microbes. Here, we investigated the ability of a bacterial isolate from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, to grow on an LMWPE model substrate for partially depolymerized and oxidized PE. Comparative genomic analyses showed that ASF-10 has a smaller genome than other Rhodococcus species yet retaining conserved functions including those related to utilization of medium- and long-chain hydrocarbons. In-depth characterization of the substrate following growth with ASF-10 confirmed depletion of alkanes and 2-ketones deriving from LMWPE, while the polymeric component remained unchanged. Proteomic analysis identified multiple enzymes that were likely to be involved in the degradation of LMWPE-derivatives, including an alkane 1-monooxygenase, cytochrome P450 hydroxylases and Baeyer-Villiger monooxygenases, as well as proteins for production of biofilm and a surfactant that may enhance accessibility to the substrate. Collectively, our findings advance the understanding of the ecology and enzymatic mechanisms underlying utilization of medium- to long-chain alkanes and oxidized variants thereof, that resemble molecules that can occur from abiotic PE degradation, by a fish gut-associated microbe. This metabolic capacity could be harnessed to develop sustainable strategies for bioremediation of oxidized, LMWPE-derivatives. ImportanceThe widespread presence of plastics in marine and freshwater environments has raised concerns due to their toxicity when ingested by fish. Microbial mechanisms driving breakdown of microplastic components, such as LMWPE and derivatives, in gut systems remain poorly understood. This study reveals how a bacterium isolated from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, metabolizes alkanes and oxidized variants thereof, that can result from abiotic PE decomposition. We identified key enzymes that are potentially involved in this process as well as in the production of biofilm and surfactants that may facilitate access to the substrate. Besides extending the knowledge of the enzymatic basis for degradation of PE-derivatives in gut-associated microbes from aquatic organisms, our results provide a framework that couples advanced compositional characterization of the substrate with omics techniques, offering valuable insight to support future studies aimed at unequivocally identifying microbes and their enzymes implicated in transformation of PE-derivatives.

microbiology↗

Microbial degradation of a widely used model polyethylene is restricted to medium- and long-chain alkanes and their oxidized derivatives

Plastics are widely used materials, yet their chemical stability hinders biodegradation, exacerbating pollution on a global scale. Soils contaminated with plastic may foster microbes adapted to degrade plastics or plastic derivatives, and these organisms and their enzymes offer promising avenues for the development of biotechnological recycling strategies. Here, two microbial communities originating from soil collected at a plastic-contaminated landfill in Norway were enriched to select for bacteria involved in the decomposition of a commonly used, model polyethylene (PE; weight average molecular weight (Mw) [~]4000 g/mol). We leveraged genome-resolved metatranscriptomics to identify active population affiliated with Acinetobacter guillouiae and Pseudomonas sp., showing a suite of upregulated genes (including those encoding alkane 1-monooxygenases, flavin-containing monooxygenases FMOs, cytochrome P450 monooxygenases) with functions compatible with degradation of oxidized products as well as medium- and long-chain hydrocarbons. Strikingly, spectroscopic, spectrometric and chromatographic analyses revealed the unexpected presence of medium- and long-chain alkanes and 2-ketones in the model PE substrate, preventing the erroneous conclusion that the community was interacting with the polymeric component. Consistently, only alkanes and 2-ketones with chain length of 10-35 were selectively degraded by an A. guillouiae isolate, as confirmed by proteomics analyses and substrate characterization following bacterial growth. Besides extending the knowledge on the enzymatic basis for degradation of PE-derivatives in soil-associated microbial systems, our results provide an advanced compositional characterization of a widely used model "PE" material, while offering valuable insight to support future studies aimed at unequivocally identifying organisms and their enzymes implicated in PE transformation.

microbiology↗

Production and evaluation of fluorophore-doped polymer substrates to screen for plastic-degrading enzymes

Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. While liquid and gas chromatography with mass spectrometry detection offer great power, the cost of the equipment is high, and the methods may not be straightforward when studying crude environmental samples. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent probe, rhodamine 6G, and we evaluate this screening method using poly(ethylene terephthalate) (PET) as a model material. Our results indicate that enzymatic depolymerization of the rhodamine-doped PET can be observed in a high-throughput fashion by following release of the fluorophore. The fluorescence data obtained during the hydrolysis of rhodamine-doped PET by 14 engineered PET hydrolases, produced with a robotic platform, correlated with the quantitative chromatographic analysis of PET degradation products. Remarkably, the use of the rhodamine-loaded PET substrate resulted in negligibly low background signals even when detecting PETase activity in crude cell lysates, suggesting suitability for screening of a wide variety of samples. Encouraged by these results, we next produced a selection of polyethylene- and nylon-based materials loaded with rhodamine 6G. While rapid leaching of fluorophore observed with nylon substrates limits the utility of the method for detecting nylonase activity, the rhodamine-loaded polyethylene showed promising performance in passive diffusion tests, indicating that this latter substrate may be used to screen for polyolefin-degrading enzymes.

biochemistry↗

Revisiting the activity of two poly(vinyl chloride)- and polyethylene-degrading enzymes

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. We attempted to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called "Ceres") or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments with the recombinant proteins, we did not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted, leading to the erroneous claim that enzymatic deconstruction of polyethylene and poly(vinyl chloride) had occurred. Our results should lead to caution when interpreting the growing amount of literature claiming enzymatic degradation of non-hydrolyzable plastics.

biochemistry↗