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1. Environmental Consortium Containing Pseudomonas and Bacillus Species Synergistically Degrades Polyethylene Terephthalate Plastic

   https://doi.org/https://doi.org/10.1128/mSphere.01151-20.  

   Cameron Roberts ; Sabrina Edwards ; Morgan Vague ; Rosa León-Zayas ; Henry Scheffer ; Gayle Chan ; Natasja A. Swartz ; Jay L. Mellies ( September 2022 , mSphere)

   Katherine McMahon, University of (Ed.)

   Plastics, such as polyethylene terephthalate (PET) from water bottles, are polluting our oceans, cities, and soils. While a number of Pseudomonas species have been described that degrade aliphatic polyesters, such as polyethylene (PE) and polyurethane (PUR), few from this genus that degrade the semiaromatic poly- mer PET have been reported. In this study, plastic-degrading bacteria were isolated from petroleum-polluted soils and screened for lipase activity that has been associ- ated with PET degradation. Strains and consortia of bacteria were grown in a liquid carbon-free basal medium (LCFBM) with PET as the sole carbon source. We moni- tored several key physical and chemical properties, including bacterial growth and modi!cation of the plastic surface, using scanning electron microscopy (SEM) and attenuated total re"ectance-Fourier transform infrared spectroscopy (ATR-FTIR) spec- troscopy. We detected by-products of hydrolysis of PET using 1H-nuclear magnetic resonance (1H NMR) analysis, consistent with the ATR-FTIR data. The full consortium of !ve strains containing Pseudomonas and Bacillus species grew synergistically in the presence of PET and the cleavage product bis(2-hydroxyethyl) terephthalic acid (BHET) as sole sources of carbon. Secreted enzymes extracted from the full consor- tium were capable of fully converting BHET to the metabolically usable monomers terephthalic acid (TPA) andmore »ethylene glycol. Draft genomes provided evidence for mixed enzymatic capabilities between the strains for metabolic degradation of TPA and ethylene glycol, the building blocks of PET polymers, indicating cooperation and ability to cross-feed in a limited nutrient environment with PET as the sole carbon source. The use of bacterial consortia for the biodegradation of PET may provide a partial solution to widespread planetary plastic accumulation.« less
2. Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism

   https://doi.org/10.1073/pnas.2121426119  

   Kincannon, William M. ; Zahn, Michael ; Clare, Rita ; Lusty Beech, Jessica ; Romberg, Ari ; Larson, James ; Bothner, Brian ; Beckham, Gregg T. ; McGeehan, John E. ; DuBois, Jennifer L. ( March 2022 , Proceedings of the National Academy of Sciences)

   Several bacteria possess components of catabolic pathways for the synthetic polyester poly(ethylene terephthalate) (PET). These proceed by hydrolyzing the ester linkages of the polymer to its monomers, ethylene glycol and terephthalate (TPA), which are further converted into common metabolites. These pathways are crucial for genetically engineering microbes for PET upcycling, prompting interest in their fundamental biochemical and structural elucidation. Terephthalate dioxygenase (TPADO) and its cognate reductase make up a complex multimetalloenzyme system that dihydroxylates TPA, activating it for enzymatic decarboxylation to yield protocatechuic acid (PCA). Here, we report structural, biochemical, and bioinformatic analyses of TPADO. Together, these data illustrate the remarkable adaptation of TPADO to the TPA dianion as its preferred substrate, with small, protonatable ring 2-carbon substituents being among the few permitted substrate modifications. TPADO is a Rieske [2Fe2S] and mononuclear nonheme iron-dependent oxygenase (Rieske oxygenase) that shares low sequence similarity with most structurally characterized members of its family. Structural data show an α-helix–associated histidine side chain that rotates into an Fe (II)–coordinating position following binding of the substrate into an adjacent pocket. TPA interactions with side chains in this pocket were not conserved in homologs with different substrate preferences. The binding mode of the less symmetric 2-hydroxy-TPA substrate,more »the observation that PCA is its oxygenation product, and the close relationship of the TPADO α-subunit to that of anthranilate dioxygenase allowed us to propose a structure-based model for product formation. Future efforts to identify, evolve, or engineer TPADO variants with desirable properties will be enabled by the results described here.« less
3. Microbial Consortia and Mixed Plastic Waste: Pangenomic Analysis Reveals Potential for Degradation of Multiple Plastic Types via Previously Identified PET Degrading Bacteria

   https://doi.org/https://doi.org/10.3390/ijms23105612  

   Sabrina Edwards ; Rosa León-Zayas ; Riyaz Ditter ; Helen Laster ; Grace Sheehan ; Oliver Anderson ; Toby Beattie ; Jay L. Mellies ( April 2022 , International journal of molecular sciences)

   The global utilization of single-use, non-biodegradable plastics, such as bottles made of polyethylene terephthalate (PET), has contributed to catastrophic levels of plastic pollution. Fortu- nately, microbial communities are adapting to assimilate plastic waste. Previously, our work showed a full consortium of five bacteria capable of synergistically degrading PET. Using omics approaches, we identified the key genes implicated in PET degradation within the consortium’s pangenome and transcriptome. This analysis led to the discovery of a novel PETase, EstB, which has been observed to hydrolyze the oligomer BHET and the polymer PET. Besides the genes implicated in PET degradation, many other biodegradation genes were discovered. Over 200 plastic and plasticizer degradation-related genes were discovered through the Plastic Microbial Biodegradation Database (PMBD). Diverse carbon source utilization was observed by a microbial community-based assay, which, paired with an abundant number of plastic- and plasticizer-degrading enzymes, indicates a promising possibility for mixed plastic degradation. Using RNAseq differential analysis, several genes were predicted to be involved in PET degradation, including aldehyde dehydrogenases and several classes of hydrolases. Active transcription of PET monomer metabolism was also observed, including the generation of polyhydroxyalkanoate (PHA)/polyhydroxybutyrate (PHB) biopolymers. These results present an exciting opportunity for the bio-recycling of mixed plasticmore »waste with upcycling potential.« less
4. Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

   https://doi.org/https://doi.org/10.1104/pp.18.01195  

   Lucas Busta, Won Cheol ( December 2018 , Plant physiology)

   Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resultingmore »in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules. Many organisms implement specialized biochemical pathways to convert ubiquitous metabolites into bioactive chemical compounds. Since plants comprise the majority of the human diet, specialized plant metabolites play crucial roles not only in crop biology but also in human nutrition. Some asterids produce lipid compounds called polyacetylenes (for review, see Negri, 2015) that exhibit antifungal activity (Garrod et al., 1978; Kemp, 1978; Harding and Heale, 1980, 1981; Olsson and Svensson, 1996) and accumulate in response to fungal phytopathogen attack (De Wit and Kodde, 1981; Elgersma and Liem, 1989). These observations have led to the longstanding hypothesis that polyacetylenes are natural pesticides. These same lipid compounds exhibit cytotoxic activity against human cancer cell lines and slow tumor growth (Fujimoto and Satoh, 1988; Matsunaga et al., 1989, 1990; Cunsolo et al., 1993; Bernart et al., 1996; Kobaek-Larsen et al., 2005; Zidorn et al., 2005), making them important nutritional compounds. The major source of polyacetylenes in the human diet is carrot (Daucus carota L.). Carrot is one of the most important crop species in the Apiaceae, with rapidly increasing worldwide cultivation (Rubatzky et al., 1999; Dawid et al., 2015). The most common carrot polyacetylenes are C17 linear aliphatic compounds containing two conjugated carbon-carbon triple bonds, one or two carbon-carbon double bonds, and a diversity of additional in-chain oxygen-containing functional groups. In carrot, the most abundant of these compounds are falcarinol and falcarindiol (Dawid et al., 2015). Based on their structures, it has been hypothesized that these compounds (alias falcarin-type polyacetylenes) are derived from ubiquitous fatty acids. Indeed, biochemical investigations (Haigh et al., 1968; Bohlman, 1988), radio-chemical tracer studies (Barley et al., 1988), and the discovery of pathway intermediates (Jones et al., 1966; Kawazu et al., 1973) implicate a diversion of flux away from linolenate biosynthesis as the entry point into falcarin-type polyacetylene biosynthesis (for review, see Minto and Blacklock, 2008). The final steps of linolenate biosynthesis are the conversion of oleate to linoleate, mediated by fatty acid desaturase 2 (FAD2), and linoleate to linolenate, catalyzed by FAD3. Some plant species contain divergent forms of FAD2 that, instead of or in addition to converting oleate to linoleate, catalyze the installation of unusual in-chain functional groups such as hydroxyl groups, epoxy groups, conjugated double bonds, or carbon-carbon triple bonds into the acyl chain (Badami and Patil, 1980) and thus divert flux from linolenate production into the accumulation of unusual fatty acids. Previous work in parsley (Petroselinum crispum; Apiaceae) identified a divergent form of FAD2 that (1) was up-regulated in response to pathogen treatment and (2) when expressed in soybean embryos resulted in production of the monoyne crepenynate and, by the action of an unassigned enzyme, dehydrocrepenynate (Kirsch et al., 1997; Cahoon et al., 2003). The results of the parsley studies are consistent with a pathogen-responsive, divergent FAD2-mediated pathway that leads to acetylenic fatty acids. However, information regarding the branch point into acetylenic fatty acid production in agriculturally relevant carrot is still largely missing, in particular, the identification and functional characterization of enzymes that can divert carbon flux away from linolenate biosynthesis into the production of dehydrocrepenynate and ultimately falcarin-type polyacetylenes. Such genes, once identified, could be used in the future design of transgenic carrot lines with altered polyacetylene content, enabling direct testing of in planta polyacetylene function and potentially the engineering of pathogen-resistant, more nutritious carrots. These genes could also provide the foundation for further investigations of more basic aspects of plant biology, including the evolution of fatty acid-derived natural product biosynthesis pathways across the Asterid clade, as well as the role of these pathways and compounds in plant ecology and plant defense. Recently, a high-quality carrot genome assembly was released (Iorizzo et al., 2016), providing a foundation for genome-enabled studies of Apiaceous species. This study also provided publicly accessible RNA sequencing (RNA-Seq) data from diverse carrot tissues. Using these resources, this study aimed to provide a detailed gas chromatography-based quantification of polyacetylenes in carrot tissues for which RNA-Seq data are available, then combine this information with bioinformatics analysis and heterologous expression to identify and characterize biosynthetic genes that underlie the major entry point into carrot polyacetylene biosynthesis. To achieve these goals, thin-layer chromatography (TLC) was combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection to identify and quantify polyacetylenic metabolites in five different carrot tissues. Then the sequences and tissue expression profiles of potential FAD2 and FAD2-like genes annotated in the D. carota genome were compared with the metabolite data to identify candidate pathway genes, followed by biochemical functionality tests using yeast (Saccharomyces cerevisae) and Arabidopsis (Arabidopsis thaliana) as heterologous expression systems.« less
5. The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

   https://doi.org/10.1073/pnas.2023348118  

   Hanson, Andrew D. ; McCarty, Donald R. ; Henry, Christopher S. ; Xian, Xiaochen ; Joshi, Jaya ; Patterson, Jenelle A. ; García-García, Jorge D. ; Fleischmann, Scott D. ; Tivendale, Nathan D. ; Millar, A. Harvey ( March 2021 , Proceedings of the National Academy of Sciences)

   Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part’s working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100–200 enzymes each from Lactococcus lactis , yeast, and Arabidopsis . CCRs in these organisms had similar ranges (<10 3 to >10 7 ) but different median values (3–4 × 10 4 in L. lactis and yeast versus 4 × 10 5 in Arabidopsis ). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes.more »Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.« less